Search

Although the etiology of nonsyndromic forms of craniosynostosis remains uncertain, recent experiments from our laboratory have demonstrated that fetal head constraint induces cranial suture fusion in mice through a process associated with altered patterns of transforming growth factor beta (TGF-beta) isoform expression. Other recent studies have highlighted the role of secreted signaling molecules, including members of the TGF-beta superfamily and the fibroblast growth factors (FGFs), as well as their receptors, in regulating suture development and fusion. The purpose of these experiments was to examine the potential role of TGF-beta receptors and FGF receptor 2 (FGFR2) in nonsyndromic craniosynostosis by determining their temporospatial patterns of expression during development complicated by intrauterine head constraint. This study consisted of two groups of C57BI/6J mice: an experimental group subjected to intrauterine constraint and a control unconstrained group. Fetal head constraint was induced by performing uterine cerclage on day 17.5 of gestation and allowing intrauterine fetal growth to continue 24 and 48 hours beyond the normal gestational period. Control animals underwent hysterotomy on day 17.5 and the nonconstrained pups were allowed to continue intra-abdominal fetal growth 48 hours beyond normal gestation. Expression of TGF-beta receptor types I and II, and FGFR2 in the calvarial tissue was determined by immunohistochemical analysis. In the unconstrained control animals, there was minimal immunoreactivity for both TGF-beta receptors and FGFR2 within the coronal suture. After 24 hours of constraint, however, there was a marked increase in immunoreactivity of TGF-beta receptors and FGFR2 in the osteoblasts along the osteogenic fronts and in the dural cells. After 48 hours, there was continued expression of both type I and type II receptors and FGFR2 within the midsutural mesenchyme of the coronal suture, in the osteoblasts, and in the dura. The authors demonstrated substantial upregulation of TGF-beta receptor types I and II and FGFR2 in coronal sutures subjected to in utero constraint. These results suggest an important role for TGF-beta/TGF-beta receptor, and FGF/FGFR signaling in the pathogenesis of constraint-induced craniosynostosis.

The inflammatory response is marked by the release of several cytokines with multiple roles in regulating leukocyte activities, including the secretion of matrix metalloproteinases (MMPs). Although the effects of individual cytokines on monocyte MMP expression have been studied extensively, few studies have examined the influence of combinations of cytokines, which are likely present at inflammatory sites. Herein, we report our investigation of the combinatorial effects of tumor necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta on MMP-9 synthesis. We found that TGF-beta suppressed TNF-alpha-induced MMP-9 secretion by MonoMac-6 monocytic cells in a dose-dependent manner, with a maximal effect of TGF-beta observed at 1 ng/ml. Such suppression was likely regulated at the pretranslational level, because steady-state mRNA levels of TNF-alpha-induced MMP-9 were reduced by TGF-beta, and pulse-chase radiolabeling also showed a decrease in new MMP-9 protein synthesis. The suppressive effects of TGF-beta were time dependent, because short exposures to TNF-alpha before TGF-beta or simultaneous exposure to both cytokines efficiently reduced MMP-9 secretion. Expression of the tissue inhibitor of metalloproteinases (TIMP)-1 and TNF-alpha receptors was unaffected by either cytokine individually or in combination. Affinity binding with radiolabeled TGF-beta demonstrated that levels of TGF-beta receptors were not increased after preincubation with TGF-beta. Suppression of TNFalpha-induced MMP-9 secretion by TGF-beta correlated with a reduction in prostaglandin E2 (PGE2) secretion. Furthermore, the effect of TGF-beta or indomethacin on blockage of TNF-alpha-stimulated MMP-9 production was reversed by the addition of either exogenous PGE2 or the cyclic AMP (cAMP) analogue Bt2cAMP. Thus, we concluded that TGF-beta acts as a potent suppressor of TNF-alpha-induced monocyte MMP-9 synthesis via a PGE2- and cAMP-dependent mechanism. These results suggest that various combinations of cytokines that are present at inflammatory sites, as well as their balance during different stages of inflammation, may provide the signals necessary for directing MMP-mediated leukocyte activities.

Increased concentrations of TGF-beta 1 in endometriotic tissue are considered important in the pathophysiology of endometrial diseases since TGF-beta 1 may inhibit natural killer activity and induce angiogenesis and proliferation of endometrial stromal cells. In the present study we report on TGF-beta 1, IGF-1 and their receptor localization, as detected by Northern hybridization and immunohistochemistry, in ovarian endometriotic tissues removed during surgical procedures. We detected comparable expression of IGF-1 and IGF-1 receptor in the stromal and epithelial compartments, thus confirming disregulated expression of the IGF system in ovarian endometriosis. On the contrary, strongly increased TGF-beta 1 steady state level mRNA expression was detected in all endometriotic samples. In addition, we demonstrated weak TGF-beta 1 immunohistochemical expression in the epithelial lining and intense expression in the cellular stroma of ovarian endometriomas, thus suggesting that TGF-beta 1 could have an important role in the maintenance and propagation of the disease. On the basis of these preliminary results we can assume that TGF-beta 1, IGF-1 and their receptors may play an important role in the pathogenesis of endometriosis.

It has been reported that transforming growth factor (TGF)-beta, which plays an integral role in the pathogenesis of idiopathic pulmonary fibrosis (IPF), suppresses proliferation of alveolar epithelial cells in vitro. Although hyperplastic lesions of alveolar lining epithelial cells (ALECs) are characteristic pathologic features of IPF, the mechanism of their involvement in the pathogenesis has not yet been extensively studied. On the assumption that the hyperplastic ALECs have escaped from the growth-inhibitory effects of TGF-beta, we searched for mutations in the microsatellite of the TGF-beta receptor type II (T beta RII) gene. To detect a deletion in the polyadenine tract in exon 3 of the T beta RII gene, cells were isolated by microdissection from lung sections of IPF patients, and DNA was extracted from these cells and amplified by high-fidelity polymerase chain reaction. A total of 121 sites of hyperplastic ALECs from 11 IPF patients were analyzed, and a one-base-pair deletion was detected in nine sites from five patients. The mutation was also detected in smooth muscle-like cells of the thickened pulmonary artery. In some tissue areas where the deletion was detected, low T beta RII expression was confirmed by immunohistochemical staining. These data suggest that microsatellite instability in the T beta RII gene occurred in some lesions of hyperplastic ALECs in IPF, although at a low incidence, and that this genetic disorder might play a partial role in the pathologic changes of IPF.

Aims/hypothesis: Activation of the renal transforming growth factor beta (TGF-beta) axis has been suggested to play a part in the development of diabetic nephropathy by a direct stimulatory effect of hyperglycaemia or through the activation of the renin-angiotensin system. Our aim was to evaluate the involvement of the renin-angiotensin system by examining the effects of ACE-inhibition on intrarenal changes in all three TGF-beta isoforms and receptors in experimental diabetes in vivo., Methods: Immunocytochemistry, western blotting and ribonuclease protection assays were carried out for each TGF-beta isoform and receptor on kidney from non-diabetic and streptozotocin-diabetic rats after treatment with the ACE inhibitor, enalapril, for 30 days., Results: Enalapril partially prevented the renal hypertrophy and fully prevented the increase in urinary albumin excretion rate in diabetic animals. The glomerular TGF-beta Type II Receptor mRNA and protein concentrations increased over 30 days in untreated diabetic animals compared with non-diabetic controls, while enalapril-treated diabetic animals showed a normalisation of TGF-beta Type II Receptor mRNA and protein., Conclusion/interpretation: The ACE-inhibition had pronounced inhibitory effects on the increased expression of the glomerular TGF-beta Type II Receptor in the diabetic kidney required for intracellular signalling through this growth factor axis. This suggests a new mechanism of action of the ACE-inhibition in regulating the development of diabetic nephropathy.

Transforming growth factor-beta (TGF-beta) signaling proceeds from the cell membrane to the nucleus through the cooperation of the type I and II serine/threonine kinase receptors and their downstream SMAD effectors. Although various regulatory proteins affecting TGF-beta-mediated events have been described, relatively little is known about receptor interactions at the level of the plasma membrane. Caveolae are cholesterol-rich membrane microdomains that, along with their marker protein caveolin-1 (Cav-1), have been implicated in the compartmentalization and regulation of certain signaling events. Here, we demonstrate that specific components of the TGF-beta cascade are associated with caveolin-1 in caveolae and that Cav-1 interacts with the Type I TGF-beta receptor. Additionally, Cav-1 is able to suppress TGF-beta-mediated phosphorylation of Smad-2 and subsequent downstream events. We localize the Type I TGF-beta receptor interaction to the scaffolding domain of Cav-1 and show that it occurs in a physiologically relevant time frame, acting to rapidly dampen signaling initiated by the TGF-beta receptor complex.

Growth factors are biologically active mediators that bind to specific receptors on target cells and regulate genes involved in cell growth, wound healing and regeneration. The expression of these receptors is thus of fundamental importance for the response of the cells to the factors. The aim here was to examine, using immunohistochemistry and flow cytometry, the expression of growth factor receptors in normal gingiva, periodontal ligament and in cells derived from these tissues, and also in regenerated tissues following guided tissue regeneration (GTR). By immunocytochemistry platelet-derived growth factor receptor-alpha (PDGF-Ralpha) was not detected in any of the tissues, whereas the PDGF-Rbeta and transforming growth factor-beta receptor types I and II (TGF-beta RI, RII) appeared to be upregulated in regenerated tissues compared with gingival and periodontal ligament tissues. Epidermal growth factor receptor (EGF-R) was also notably elevated in the regenerated tissue and was strongly expressed in the gingival epithelium but not in the periodontal ligament. Neither were fibroblast growth factor receptor-I (FGF-RI) or insulin-like growth factor receptor (IGF-R) detected in the periodontal ligament, nor in the gingiva, but they sometimes stained weakly in the regenerated tissues. Flow cytometry (FCM) showed that all the cells derived from the normal gingiva and the periodontal ligament expressed the PDGF-Rbeta, whereas the TGF-beta RI and RII, FGF-RI and IGF-R were detected in only a proportion of the total cells. In contrast, none of the cells expressed the PDGF-Ralpha or the EGF-R. These observations show that the growth factor receptors are differentially expressed by the periodontal tissues and cells and suggest that the corresponding factors may also be differentially involved in periodontal wound healing and regeneration.

Transforming growth factor (TGF)-beta is an immunosuppressive agent that is efficacious in suppressing a wide variety of cell-mediated immune responses. However, the direct effect of this cytokine on Langerhans cells (LC) has not been clarified. In this study, we examined its modulatory effects on the expression of co-stimulatory molecules and LC IL-12 production. A highly purified population of LC (>95%) was prepared from BALB/c mouse skin by the panning method using anti-I-Ad mAb. Semiquantitative reverse transcription-PCR analysis showed that LC express TGF-beta receptor II mRNA. Interestingly, TGF-beta1 enhanced IL-12 p40 production of anti-CD40/IFN-gamma-stimulated LC, despite its down-regulatory effect on CD40 expression. A bioassay using an IL-12-dependent T cell line demonstrated the correlation of the IL-12 p40 level with the bioactivity of IL-12. More importantly, it was found that in contrast to TGF-beta, granulocyte/macrophage colony-stimulating factor (GM-CSF) strikingly inhibits IL-12 production of anti-CD40/IFN--stimulated LC and that the level of LC IL-12 production is determined by the relative amounts of TGF-beta1 and GM-CSF. Taken together, these results suggest that the two cytokines produced in the skin microenvironment, namely TGF-beta and GM-CSF, exert their important effects on LC function by regulating the secretion of IL-12, a cytokine influencing the Th1-Th2 balance.

Background: Medical treatment of benign prostatic hyperplasia (BPH) targets relief of symptoms by causing either relaxation of the prostatic smooth muscle with alpha1 adrenergic blockade, or shrinkage of the gland with 5alpha-reductase inhibitors. We recently demonstrated that alpha1-blockers, such as terazosin, induce apoptosis in prostatic cells. In this study, we examined the combined effect of finasteride and terazosin on the rate of apoptosis and cellular proliferation to investigate their potential synergy at the cellular level., Methods: Prostate specimens were obtained from men who were treated with either finasteride (n = 24), terazosin (n = 42), or combination therapy (n = 10) for varying time periods (1 week to 36 months) for the relief of the symptoms of BPH. The proliferative and apoptotic indices of both stromal and epithelial prostatic cell populations were determined. Antibodies against TGF-beta1 and TbetaRII were used to examine the immunoreactivity of TGF-beta1 and TbetaRII, respectively, in all prostate tissue sections., Results: The apoptotic index in both prostate cell populations was significantly higher following the combination treatment compared to terazosin or finasteride alone. There were no significant changes in the rate of cellular proliferation with any treatment. Furthermore, there was a significant increase in TGF-beta1 expression in the prostates of patients treated with terazosin or combination therapy, while there was no change in TbetaRII expression., Conclusions: These results support the concept that induction of prostate apoptosis is a potential molecular mechanism underlying the combination effect of alpha1 blockade with 5alpha-reductase inhibitors in the effective treatment of BPH. The upregulation of TGF-beta1 implies a role for this ligand as an effector of apoptosis induction in response to alpha1-blockade or finasteride therapy of BPH patients., (Copyright 2001 Wiley-Liss, Inc.)

Objective: To study the expression of transforming growth factor-beta receptor I (TGF-beta RI) and its mRNA in liver tissues of chronic hepatitis B (CHB) and the relationship between its expression and the pathological injury of hepatic tissues., Methods: Forty-eight liver specimens from patients with CHB were observed by techniques of immunohistochemistry and in situ nucleic acid hybridization., Results: TGF-beta RI was expressed in the hepatocyte, sinusoid endothelial cell and bile duct epithelial cell, with predominance in the hepatocyte and distribution in small granule or cell membrane. Increased expressing of TGF-beta RI was parallel to the degree of inflammatory activity and fibrosis (r=0.7095, P<0.05; r=0.6915, P<0.05; respectively). The number and the distribution of TGF-beta RI mRNA in the positive cells were coincident to those of its protein. The expression of TGF-beta RI was closely correlated with Tbil (r= 0.3396, P<0.05), PA (r= -0.3430, P<0.05) and ALB (r= -0.4173, P<0.01)., Conclusion: The TGF-beta RI may involve in the pathological injury of hepatic tissues of CHB.

Angiotensin II recruits transforming growth factor beta(1) (TGFbeta(1)) and is related to left ventricular fibrosis. However, it is unclear whether chronic in vivo reduction in left ventricular TGFbeta(1) expression blunts fibrosis and improves outcome in angiotensin II-dependent hypertension. Four-week-old male hypertensive TGR(mRen2)27 (Ren2) rats received either normal food, low-dose losartan (0.5 mg. kg(-1). d(-1)), or tranilast (a nonspecific TGFbeta inhibitor; 400 mg. kg(-1). d(-1)) (n=10 for each group) for 12 weeks and were compared with Sprague-Dawley control rats. The effect of tranilast on survival was evaluated in 34 additional untreated homozygous Ren2 rats. Tranilast or low-dose losartan did not lower blood pressure. However, the increase in left ventricular weight (Ren2 versus SD 3.1+/-0.16 versus 2.1+/- 0.06 mg/g body wt; P<0.05) was significantly (P<0.05) blunted by both tranilast (2.7+/-0.05) and losartan (2.7+/-0.07). Both drugs prevented the increase in left ventricular TGFbeta(1) mRNA and fibronectin mRNA and blunted the increase in hydroxyproline content and the increase in perivascular fibrosis. The perivascular fibrosis score correlated significantly with the level of expression of TGFbeta(1) (r=0.62; P=0.019). In situ hybridization demonstrated increases in TGFbeta(1) mRNA, predominantly in perivascular and nonmyocyte areas. Both drugs did not prevent the decrease in systolic or diastolic dP/dt, but tranilast significantly improved the survival of untreated Ren2 rats (P=0.029). In conclusion, TGFbeta(1) mRNA expression is increased predominantly in nonmyocyte regions in the hypertrophied left ventricle in this angiotensin II-dependent model of hypertension. This increase is probably due to high angiotensin II levels rather than to hypertension. This is the first study to suggest that chronic inhibition of TGFbeta(1) expression attenuates left ventricular hypertrophy and fibrosis, even without lowering blood pressure.

Accumulating evidence suggests the involvement of TGF-beta in the process of corneal opacity, which is one of the serious causes of visual loss. However, whether TGF-beta is indeed critical for the pathogenesis remains unknown. We constructed an adenovirus expressing an entire ectodomain of the human type II TGF-beta receptor fused to Fc portion of human IgG (AdTbeta-ExR): this soluble receptor is secreted from AdTbeta-ExR-infected cells, binds to TGF-beta and inhibits TGF-beta signaling. When AdTbeta-ExR was injected into the femoral muscle of Balb/c mice, a high level of the soluble receptor protein (2.0-3.5 x 10(3) pM) was detectable in the serum and in the ocular fluid for at least 10 days. In the mice subjected to corneal injury with silver nitrate and to intramuscular injection with either saline or a control adenovirus expressing beta-galactosidase (AdLacZ), corneal opacification composed of extracellular matrix (ECM) accumulation, of infiltration of neutrophils and monocytes/macrophages, and of angiogenesis were all induced. In contrast, they were markedly reduced in the mice injected with AdTbeta-ExR. Immunohistochemical analysis revealed that TGF-beta, fibronectin, macrophage chemoattractant protein-1, and vascular endothelial growth factor were densely stained in the edge of wounded cornea, but they were scarcely present in the injured-cornea of AdTbeta-ExR-treated mice. Our results demonstrate that TGF-beta indeed plays a critical role in the process of cornea opacification, and that adenovirus-mediated expression of a soluble TGF-beta receptor can be therapeutically useful.

Background: Several studies have suggested that transforming growth factor-beta1 (TGF-beta1) is an important determinant of diabetic glomerular injury. TGF-beta1 forms a heteromeric complex with two cellular receptor subtypes, designated TGF-beta RII and TGF-beta RI, but the effects of diabetes mellitus on glomerular TGF-beta receptor expression have not been completely elucidated. We first compared the effect of experimental type I diabetes mellitus and uninephrectomy on glomerular TGF-beta receptor expression in spontaneously hypertensive rats (SHRs), and then sought to determine whether changes in TGF-beta receptor expression were strain specific by studying normotensive Wistar-Kyoto (WKY) rats., Methods: Five groups of male SHRs were studied. The first group received streptozotocin (60 mg/kg IV) and was studied after one week. The second group received streptozotocin and was studied after two weeks. The third group received streptozotocin (60 mg/kg IV) but received insulin to maintain euglycemia. The fourth group of age-matched SHRs served as the control group, while a fifth group of SHRs underwent uninephrectomy. Four groups of male WKY rats were also studied. The first group of WKY rats served as the age-matched control group. The second group of WKY rats received streptozotocin, while a third group of WKY rats underwent uninephrectomy. The fourth group underwent uninephrectomy and received streptozotocin. At each time point, glomeruli were isolated for protein extraction, and the protein was subjected to Western blot analysis of TGF-beta RII and TGF-beta RI expression., Results: Basal expression of both TGF-beta receptors per microgram of glomerular protein was similar in normotensive WKY rats and hypertensive SHRs. Hyperglycemia (blood glucose level, 17.8 +/- 2.9 mmol/L) led to an early twofold increase in TGF-beta RII protein expression and a fourfold increase in TGF-beta RI protein expression in the glomeruli of hypertensive diabetic SHRs compared with euglycemic SHRs (blood glucose level, 5.8 +/- 0.8 mmol/L), which was sustained after two weeks. Insulin treatment (blood glucose level, 5. 2 +/- 0.9 mmol/L) normalized both TGF-beta RII and TGF-beta RI expression in the glomeruli of SHRs that received streptozotocin. Glomerular capillary hypertension in the uninephrectomized SHRs led to a twofold increase in glomerular TGF-beta RII protein expression, but did not reproduce the effect of diabetes mellitus on TGF-beta RI expression. In contrast to the findings in SHRs, neither hyperglycemia (blood glucose level, 15.5 +/- 2.1 mmol/L), uninephrectomy, nor hyperglycemia (blood glucose level, 16.8 +/- 3.0 mmol/L) and uninephrectomy altered TGF-beta receptor expression in the glomeruli of normotensive WKY rats., Conclusion: These studies support the hypothesis that hemodynamic factors and metabolic factors influence glomerular TGF-beta receptor in vivo in the SHRs.

A large body of evidences implicates transforming growth factor-beta (TGF-beta) in the pathogenesis of atherosclerosis. In this context, TGF-beta receptor dysfunction has been suggested to be relevant. We tested the effect of hypercholesterolemia, a well-known risk factor for atherosclerosis, on liver type II TGF-beta receptor (TbetaR-II) expression in atherosclerosis-susceptible C57BL/6 mouse strain fed atherogenic diet. In addition, the relationship between cholesterol and TbetaR-II expression was verified by cholesterol challenge on human hepatoma cell (HepG2) cultures. The susceptible C57BL/6 mice fed atherogenic diet exhibited significant mRNA and immunohistochemical TbetaR-II liver expression at 2, 5, 9 and 15 weeks as compared to animals fed a regular diet. The TbetaR-II profile on HepG2 resulted in a time-dependent increased expression when the cells were incubated with soluble free cholesterol, associated with an increased TGF-beta-dependent biological activity as detected by luciferase assay of reporter gene. These data provide evidence for a cholesterol-dependent TbetaR-II induction that may play a potentially relevant role in the development of hypercholesterolemia and atherogenesis.

Objective: To evaluate the factors involved in bone remodeling and wound healing that may be altered by radiation therapy., Design: A prospective, controlled study of biochemical activity in vitro., Subjects: MC3T3-E1 mouse osteoblasts., Interventions: Cells were irradiated at 0, 2, 4, or 6 Gy. Specimens were harvested at 1, 7, 14, 28, and 42 days following irradiation for immunohistochemical analysis of transforming growth factor beta(1) expression and transforming growth factor beta(1) type I and II receptor expression. Collagen production was measured at 1, 7, 28, 35, and 49 days after irradiation. The effects of dexamethasone on collagen production and cell proliferation were also examined., Results: Irradiated cells demonstrated decreased cell proliferation and a dose-dependent, sustained reduction in collagen production when compared with control cells. An increase in transforming growth factor beta(1) type I and II receptor expression was noted in irradiated cells when compared with controls., Conclusion: Radiation-induced alterations of factors related to bone remodeling and wound healing have a potential role in the pathogenesis of osteoradionecrosis.

The temporal and spatial immunolocalization of TGF-betas and their receptors in the intervertebral disk of senescence-accelerated mouse (SAM) was examined to determine the biological roles played by TGF-betas and their receptors in the process of intervertebral disk degeneration. Ten male SAM and ICR mice aged 8, 24 or 50 weeks after birth were used for this experiment. Histological and immunohistochemical studies using specific antibodies for TGF-beta1, -beta2, -beta3, TbetaR-I, and TbetaR-II were performed. Intervertebral disks of SAM exhibited more degenerative changes than those of ICR mice. Expression of TGF-betas and TbetaRs in disk of SAM and ICR mice was observed at 8 weeks of age, and became weaker with aging. Our results suggest TGF-betas may play a role in the growth and maintenance of intervertebal disks.

We investigated whether anti-transforming growth factor beta (TGF-beta) molecular intervention can halt the progression of liver fibrosis in rats. To block TGF-beta action in a specific manner, we prepared an adenovirus expressing a truncated type II TGF-beta receptor (AdTbeta-TR), which specifically inhibits TGF-beta signaling as a dominant-negative receptor. We also used an adenovirus expressing bacterial beta-galactosidase (AdLacZ) as a control adenovirus. Rats were treated with dimethylnitrosamine (DMN) for 3 weeks; then, AdTbeta-TR, AdLacZ, or saline was intravenously applied once, followed by an additional 3-week DMN treatment. The ratio between the truncated receptor and the wild-type receptor at the mRNA level was 15 at 1 week and 10 at 3 weeks after gene transfer. Immunohistostaining analysis showed that the truncated receptor was expressed mainly in septal cells including hepatic stellate cells. Liver fibrosis, as assessed by histology, hydroxyproline content, and the serum level of hyaluronic acid, progressed during the additional 3-week DMN treatment. However, in rats infected with AdTbeta-TR, the fibrosis remained at the level seen in rats given DMN for only 3 weeks. All AdTbeta-TR-treated rats remained alive, whereas DMN-treated rats infused with either AdLacZ or saline died of liver dysfunction. In the livers of AdTbeta-TR-treated rats, electron microscopy showed: 1) less accumulation of extracellular matrix proteins in the Disse's spaces; 2) regenerated hepatocytes; and 3) fat droplet-rich "quiescent" hepatic stellate cells. Our results demonstrate that TGF-beta plays a critical role in the progression of liver fibrosis, and suggest that anti-TGF-beta intervention should be therapeutic in already-established fibrotic livers, not only by suppressing fibrosis, but by facilitating hepatocyte regeneration.

Transforming growth factor-beta (TGF-beta) signaling requires the functional interaction of two distinct receptors, type I (RI) and type II (RII), at the cell surface. Exposure of cells to TGF-beta results in receptor internalization and down-regulation (Zwaagstra et al., 1999, Exp. Cell Res. 252, 352362); however, little is known about the subsequent fate of RI or RII. In this study the cellular distribution of RI was examined in cells before and after treatment with ligand. RI was localized by immunocytochemistry and confocal microscopy using two polyclonal antisera directed against two different epitopes, one in the C-terminal region and one in the N-terminal region of the cytoplasmic domain. The majority of RI molecules in untreated MvlLu and A549 cells were found to be intracellular. Treatment of MvlLu and A549 cells with 100 pM TGF-beta1 for 24 h at 37 degrees C caused a redistribution of surface RI on MvlLu cells, as evidenced by surface RI aggregation. Unexpectedly, this TGF-beta1 treatment also caused redistribution and accumulation of intracellular RI in and around the nucleus for both MvlLu and A549 cells. Nuclear accumulation of RI was also promoted independently of ligand receptor activation by treatment of MvlLu cells with olomoucine, an agent that results in growth arrest. The capacity of RI to localize in the nucleus was confirmed by microscopic examination of 293 cells transiently expressing RI fused to green fluorescent protein (RI-GFP). Olomoucine treatment of these cells resulted in the movement of RI-GFP into the nucleus. Our results indicate that growth arrest alters intracellular transport/routing of RI and may indicate that RI functions not only at the cell surface but inside the cell as well.

Background: Diabetes mellitus-induced nephromegaly is thought to involve both hyperplastic and hypertrophic proximal tubule cell growth. The temporal relationship between these growth patterns and the mechanisms that mediate them are unknown., Methods: Renal growth was assayed in isolated renal proximal tubules harvested from diabetic rats. Diabetes mellitus was induced by streptozotocin., Results: Following the induction of a diabetic state, there was a progressive increase in the kidney:body weight ratio. This was associated with an increase in 5-bromo-2-deoxyuridine incorporation (marker for hyperplastic cell growth) at day 2, which returned to baselines levels by day 4, and an increase in the protein:DNA ratio (marker for hypertrophic cell growth), which was clearly evident by day 10. Thus, diabetes-induced proximal tubule growth involved an initial hyperplastic, followed by a hypertrophic, growth period. During the hyperplastic growth period, both cdk4/cyclin D (cyclin D) and cdk2/cyclin E (cyclin E) kinase activities were increased. The switch between the growth periods was associated with continued activation of cyclin D, but inhibition of cyclin E kinase. The reduction in cyclin E kinase activity correlated with a reduction in cdk2/cyclin E complex abundance and an increased abundance of cyclin kinase inhibitors in cdk2/cyclin E complexes that did form. Also associated with the switch in growth patterns was a change in transforming growth factor-beta (TGF-beta) receptor expression. During the hyperplastic growth period, TGF-beta receptor II expression was decreased, while during the hypertrophic growth period, there was both a return of receptor II expression to baseline levels and increased expression of receptor I. Consistent with an increase in TGF-beta signaling during hypertrophy, there was an increase in Smad 2/3 protein expression and an increase in the abundance of Smad 2/4 complexes., Conclusions: Diabetes-induced proximal tubule growth involves an initial hyperplastic growth period that switches to a hypertrophic growth period within a couple of days. The pattern of G1 kinase activity associated with the growth pattern switch demonstrates that the hypertrophy is mediated by a cell cycle-dependent mechanism. Regulation of TGF-beta receptor expression and signaling activity through the Smad protein cascade possibly plays a role in the growth pattern switch.

Two prostatic epithelial lines, one of basal origin and one of luminal origin, were established from the dorsolateral prostates of p53 null mice. The cell lines are nontumorigenic when inoculated subcutaneously under the renal capsule or intraprostatically in syngeneic mice. The luminal cell line (PE-L-1) expresses cytokeratins 8 and 18 and the basal cell line (PE-B-1) expresses cytokeratins 5 and 14. The basal cells require serum for growth, whereas the luminal cells grow only in serum-free medium. Both cell lines require the presence of growth factors for optimal growth in culture, with EGF and FGF-2 having the greatest effect on the growth rate. Both lines express androgen receptor (AR) mRNA and protein. Androgen stimulates growth of the basal cell line, indicating that the ARs are functional, whereas growth of the luminal cells is unaffected by androgens. The luminal line is significantly inhibited by exogenous TGF-beta and produces low levels of endogenous TGF-beta. In contrast, the basal cell line produces significant amounts of TGF-beta and its growth is not influenced by this cytokine. Coculture of luminal cells with prostatic smooth muscle cells results in the generation of increased levels of biologically active TGF-beta, indicating a paracrine mechanism of TGF-beta activation that may be involved in the maintenance of normal prostatic function. To our knowledge this is the first report describing both basal and luminal prostatic cell lines from a single inbred animal species and the first indication that prostatic epithelial and stromal cells interact to generate the biologically active form of TGF-beta. These lines will provide an important model for determining basal/luminal interactions in both in vitro and in vivo assays., (Copyright 2000 Wiley-Liss, Inc.)

The steroidogenic activity of testicular Leydig cells is controlled both by the pituitary hormone (LH) and by growth factors such as transforming growth factor-beta peptides (TGFbeta1, -2, and -3; inhibin/activin; and anti-Mullerian hormone). By using primary cultures of porcine Leydig cells as a model, the aim of the study was to identify and characterize the TGFbeta receptors and to study their regulation by LH/hCG. TGFbeta receptors have been identified and characterized through three different approaches, including cross-linking experiments and Western and Northern blotting analyses. In cross-linking experiments, labeled TGFbeta was shown to bind to three different molecular species of 300, 80, and 53 kDa, which may correspond to the protein betaglycan (also known as TGFbeta type III receptor) and TGFbeta type II and I receptors (TGFbetaRII and TGFbetaRI), respectively. The presence of TGFbetaRI and -RII was further demonstrated by Western blotting analysis using specific polyclonal antibodies. Finally, the expression of betaglycan, TGFbetaRII, and TGFbetaRI messenger RNAs, was confirmed by Northern blotting analysis, as shown by the presence of 6.4-, 4.6-, and 5.8-kb messenger RNAs, respectively. By using a RT-PCR approach, the mediators of the TGFbeta signal, Smads 1-7, were also detected in cultured Leydig cells. TGFbetaRI and TGFbetaRII protein levels were enhanced by hCG/LH in a dose-dependent (maximal effect with 0.3 ng/ml hCG) and time-dependent (maximal effect observed after 48 h of hCG treatment) manner. Furthermore, to determine whether the stimulatory effect of LH/hCG was mediated by testosterone, use was made of aminogluthetimide, an inhibitor of cytochrome P450scc. The inhibition oftestosterone formation did not affect the stimulatory effect of LH/hCG on TGFbetaRI and -RII levels, suggesting that the gonadotropin action is not mediated by the steroid hormone. Together, the present findings demonstrate that the TGFbeta receptors are expressed and are under hormonal (gonadotropin) control in cultured porcine Leydig cells.

Objective: The association of transforming growth factor-beta 1 (TGF-beta 1) with a matrix metalloproteinase (MMP) and a tissue inhibitor of metalloproteinase (TIMP), as well as myometrial invasion of endometrial cancer was studied., Methods: The effects of TGF-beta 1 on cellular invasiveness, gelatinase activity, and expression of TIMP-1 were examined in 2 endometrial adenocarcinoma cell lines, KLE and Ishikawa. Plasma was obtained from 8 endometrial cancer patients with Stage-Ia disease, from 6 with Stage-Ib disease, and from 4 with Stage-Ic disease, and the levels of TGF-beta 1 were measured by enzyme immunoassays. The immunohistochemical expression of MMP-9, TIMP-1, TGF-beta 1, and TGF-beta receptor Type I in tumor tissue from the same patients also was detected., Results: Invasiveness, gelatinase activity, and the expression of TIMP-1 were higher in KLE cells than in Ishikawa cells, and they were increased by treatment with rTGF-beta 1. The expression of TGF-beta receptor Type I was higher in KLE cells than in Ishikawa cells, which were unresponsive to exogenous TGF-beta 1. The plasma levels of TGF-beta 1 were greater in Stage-Ib and Stage-Ic patients than in Stage-Ia patients. MMP-9 and TGF-beta receptor Type I were expressed mainly in tumor cells, while TIMP-1 and TGF-beta 1 were localized in both tumor epithelial cells and stromal cells. MMP-9 and TIMP-1 were expressed only in Stage-Ib and Stage-Ic patients, although TGF-beta 1 and TGF-beta receptor Type I were ubiquitous., Conclusions: Myometrial invasion of endometrial cancers involves an increase in gelatinase activity, regulated to some extent by TGF-beta 1 in an autocrine or paracrine fashion.

Poorly healing mandibular fractures and osteotomies can be troublesome complications of craniomaxillofacial trauma and reconstructive surgery. Gene therapy may offer ways of enhancing bone formation by altering the expression of desired growth factors and extracellular matrix molecules. The elucidation of suitable candidate genes for therapeutic intervention necessitates investigation of the endogenously expressed patterns of growth factors during normal (i.e., successful) fracture repair. Transforming growth factor beta1 (TGF-beta1), its receptor (Tbeta-RII), and the extracellular matrix proteins osteocalcin and type I collagen are thought to be important in long-bone (endochondral) formation, fracture healing, and osteoblast proliferation. However, the spatial and temporal expression patterns of these molecules during membranous bone repair remain unknown. In this study, 24 adult rats underwent mandibular osteotomy with rigid external fixation. In addition, four identically treated rats that underwent sham operation (i.e., no osteotomy) were used as controls. Four experimental animals were then killed at each time point (3, 5, 7, 9, 23, and 37 days after the procedure) to examine gene expression of TGF-beta1 and Tbeta-RII, osteocalcin, and type I collagen. Northern blot analysis was used to compare gene expression of these molecules in experimental animals with that in control animals (i.e., nonosteotomized; n = 4). In addition, TGF-beta1 and T-RII proteins were immunolocalized in an additional group of nine animals killed on postoperative days 3, 7, and 37. The results of Northern blot analysis demonstrated a moderate increase (1.7 times) in TGF-beta1 expression 7 days postoperatively; TGF-beta1 expression returned thereafter to near baseline levels. Tbeta-RII mRNA expression was downregulated shortly after osteotomy but then increased, reaching a peak of 1.8 times the baseline level on postoperative day 9. Osteocalcin mRNA expression was dramatically downregulated shortly after osteotomy and remained low during the early phases of fracture repair. Osteocalcin expression trended slowly upward as healing continued, reaching peak expression by day 37 (1.7 times the control level). In contrast, collagen type IalphaI mRNA expression was acutely downregulated shortly after osteotomy, peaked on postoperative days 5, and then decreased at later time points. Histologic samples from animals killed 3 days after osteotomy demonstrated TGF-beta1 protein localized to inflammatory cells and extracellular matrix within the fracture gap, periosteum, and peripheral soft tissues. On postoperative day 7, TGF-beta1 staining was predominantly localized to the osteotomized bone edges, periosteum, surrounding soft tissues, and residual inflammatory cells. By postoperative day 37, complete bony healing was observed, and TGF-beta1 staining was localized to the newly formed bone matrix and areas of remodeling. On postoperative day 3, Tbeta-RII immunostaining localized to inflammatory cells within the fracture gap, periosteal cells, and surrounding soft tissues. By day 7, Tbeta-RII staining localized to osteoblasts of the fracture gap but was most intense within osteoblasts and mesenchymal cells of the osteotomized bone edges. On postoperative day 37, Tbeta-RII protein was seen in osteocytes, osteoblasts, and the newly formed periosteum in the remodeling bone. These observations agree with those of previous in vivo studies of endochondral bone formation, growth, and healing. In addition, these results implicate TGF-beta1 biological activity in the regulation of osteoblast migration, differentiation, and proliferation during mandibular fracture repair. Furthermore, comparison of these data with gene expression during mandibular distraction osteogenesis may provide useful insights into the treatment of poorly healing fractures because distraction osteogenesis has been shown to be effective in the management of these difficult clinical cases.

Liver cancer and gastric cancer are the most common solid tumors worldwide. Transforming growth factor-beta (TGF-beta) production and lack of response to TGF-beta growth inhibitory effects have been associated with tumor progression and therapeutic resistance. HepG2, Hep3B, and SK-HEP-1 human liver cancer lines produce 3, 5.7, and 2.5 ng TGF-beta1; 1.4, 2, and 4 ng TGF-beta2 and 0.15, 0.2 and 0.22 ng TGF-beta3 per 107 cells (24 h). Expression of the TGF-beta type I receptor is 20x, 1x, and 0.6x the level in mink lung MvLu1 cells in the HepG2, Hep3B, and SK-HEP-1 cells, respectively. HepG2 and Hep3B cells do not express the TGF-beta type II receptor while SK-HEP-1 cells express 7x the level found in mink lung MvLu1 cells. Hs 746T, KATO III, RF-1, and RF-48 human gastric cancer cell lines produce 12. 5, 0.35, 0.4, and 0.4 ng TGF-beta1; 2.6, 0.95, 0.5, and 0.52 ng TGF-beta2 and 0.42, 0.17, 0.12, and 0.14 ng TGF-beta3 per 107 cells (24 h). Expression of TGF-beta type I receptor is 0.7x, 0.7x, 0.8x, 0.6x the level in mink lung MvLu1 cells in the Hs 746T, KATO III, RF-1 and RF-48 cells, respectively. KATO III cells are lacking in the TGF-beta type II receptor while Hs 746T, RF-1 and RF-48 cells express 10x, 0.8x, and 1x the levels in mink lung MvLu1 cells. The IC50 for TGF-beta1 is >>10 ng/ml in all of these lines except RF-48 where TGF-beta1 is mitogenic. The response of the cell lines to radiation, doxorubicin, mitomycin C, cisplatin, 5-fluorouracil, methotrexate, and gemcitabine showed that SK-HEP-1 was the most drug resistant liver cancer cell line and KATO III was the most drug resistant gastric cancer cell line. Overall, there was no correlation between TGF-beta secretion in cell culture and sensitivity of the cells to anticancer agents. Increased TGF-beta1 levels were detectable in the plasma of nude mice bearing Hep3B and Hs 746T xenografts. Those tumors which secreted greater amounts of TGF-beta were more therapeutically resistant in vivo.

Background: Considerable controversy exists regarding impairment of cardiac function in diabetes mellitus (DM). We investigated the serial changes in left ventricular (LV) histopathology and LV filling dynamics in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, which have been established as an animal model of type II DM., Methods and Results: In 54 OLETF and 54 non-DM rats, body weight, blood pressure, heart rate, and transmitral pulsed Doppler examinations were performed from 5 to 47 weeks of age. An oral glucose tolerance test was performed at 10, 20, and 30 weeks of age. The hearts were excised for histopathology, including immunohistochemistry and histomorphometry of collagen, and measurement of hydroxyproline at baseline and each stage of developing DM. In the prediabetic stage (15 weeks of age), in which fast blood glucose remained normal, OLETF rats manifested mild obesity, postprandial hyperglycemia, and hyperinsulinemia, and early diastolic transmitral inflow exhibited prolonged deceleration time (OLETF, 59+/-10 ms versus non-DM, 49+/-8 ms, P<0.01) and low peak velocity (OLETF, 73+/-11 cm/s versus non-DM, 88+/-11 cm/s, P<0.01). Histopathology revealed extracellular fibrosis and abundant transforming growth factor-beta(1) receptor II in LV myocytes of OLETF rats. At 15 weeks of age, the ratio of collagen area/visual field of LV wall in OLETF rats (8.3+/-1.3%) was larger than that in non-DM rats (4.9+/-1.8%, P<0.0001), and the collagen content/dry tissue weight ratio of heart was significantly higher in OLETF (2. 0+/-0.5 mg/g) than non-DM (1.3+/-0.2 mg/g, P<0.01) rats., Conclusions: A metabolic abnormality present in the prestage of type II DM may produce LV fibrosis and alteration in cardiac function.

Aims: Loss of transforming growth factor beta type II receptor (TGFbeta-RII) expression has been associated with resistance to TGFbeta-mediated inhibition of cell proliferation and tumour progression. We investigated whether the expression of TGFbeta-RII is related to the progression of human breast cancer and whether there is a correlation between TGFbeta-RII expression and phenotypic markers of biological aggressiveness., Methods and Results: Immunohistochemical methods were used to detect TGFbeta-RII in archival breast samples including benign proliferative lesions, ductal carcinoma in situ (DCIS) and invasive mammary carcinomas (IMC). Neoplastic cells showed reduced expression of TGFbeta-RII in comparison to the normal breast tissue and benign lesions. There was a significant inverse correlation between loss of TGFbeta-RII expression and tumour grade within both DCIS (P = 0.004) and IMC (P = 0.001) groups. There was an inverse correlation between TGFbeta-RII expression and both mitotic count (P = 0.001) and clinical stage (P = 0.004). Oestrogen receptor (P = 0.07) and lymph node status (P = 0.10) were not significantly associated with TGFbeta-RII expression., Conclusions: These data indicate that decreased expression of TGFbeta-RII may contribute to breast cancer progression and is related to a more aggressive phenotype in both in-situ and invasive carcinomas.

The dystrophin-deficient mdx mouse presents muscle fiber necrosis but active muscle regeneration, probably due to an extensive recruitment of myogenic regulatory factors (MRF), several growth factors and cytokines, and favorable interaction of satellite cells. In contrast, the laminin alpha 2 (merosin)-deficient dy mouse shows progressive muscle fiber necrosis and ineffective muscle regeneration. Using Western blot and immunohistochemical analyses, we investigated the adaptive changes in MRF, growth factors and cytokines and their receptors in the muscles of dy mice during postnatal growth. The relative volume of MyoD, myogenin and Myf-5 proteins was markedly lower in the gastrocnemius and rectus femoris muscles of dy mice. Transforming growth factor-beta 2, leukemia inhibitory factor (LIF) and basic fibroblast growth factor were not up-regulated in the muscles of dy mice. The levels of the LIF receptor and insulin-like growth factor-I receptor levels were markedly decreased in the muscles of dy mice during the entire postnatal period observed in this study. Therefore, unlike the situation in mdx mice, the milieu of regeneration following repetitive damage seems to be degraded in the muscles of dy mice.

Background: Advanced glycation end product-modified beta2-microglobulin (AGE-beta2m) is an important component of dialysis-related amyloidosis (DRA). Its presence induces monocyte chemotaxis and the release of the proinflammatory cytokines through macrophage activation. Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine that also has chemotactic activity for monocytes at very low (0.1 to 10 pg/mL) concentrations and inhibits proinflammatory cytokine production of macrophages. In this study, we investigated the role of TGF-beta in the pathogenesis of DRA., Methods: We performed an immunohistochemical study of DRA tissues (8 cases) to confirm the existence of TGF-betas and their receptors; we also performed a chemotaxis assay of human monocytes as well as enzyme-linked immunosorbent assay (ELISA) of TGF-beta1, tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interleukin-1 receptor antagonist (IL-1Ra) in the supernatant of human monocyte-derived macrophage cell culture under varying conditions of incubation with TGF-beta1, AGE-beta2m, and TGF-beta1 antibody additions., Results: There was positive staining for TGF-betas (types 1, 2, and 3) and their receptors (types I, II, and III) in infiltrated macrophages (CD68+), synovial lining cell, as well as vascular walls around amyloid deposition. AGE-beta2m also induced TGF-beta1 production by macrophages in a dose-dependent manner (410 +/- 80 pg/mL at 12.5 microg/mL, 621 +/- 62 pg/mL at 25 microg/mL, and 776 +/- 62 pg/mL at 50 microg/mL of AGE-beta2m). AGE-beta2m induced significant TNF-alpha and IL-1Ra production by macrophage. The addition of exogenous TGF-beta1 (0.1 to 10 ng/mL) decreased AGE-beta2m-induced TNF-alpha production and increased IL-1Ra production in a dose-dependent fashion. IL-1beta production was not effected by any experimental conditions. In chemotaxis assay, anti-TGF-beta1 antibody (0.1 to 10 microg/mL) attenuated AGE-beta2m-induced monocyte chemotaxis., Conclusions: These results provide the first evidence to our knowledge for the presence of TGF-beta in DRA tissue, as well as the stimulatory action of AGE-beta2m on tissue macrophages. In turn, TGF-beta suppresses the proinflammatory activation of macrophages, suggesting a dual role for TGF-beta in the inflammatory process of DRA. These observations may provide a pathophysiologic link between TGF-beta and DRA.

Using a muscle cell line and satellite cell cultures, it has been shown that transforming growth factor-beta (TGF-beta) has a powerful inhibitory effect on myoblast replication and differentiation. However, little work has been done on the possible role of TGF-beta in adult muscle in vivo. Using Western blot and immunohistochemical analyses, we investigated normal distribution of TGF-beta 2 and TGF-beta RII proteins between slow and fast-type muscles, and the adaptive response of these proteins in the mechanically overloaded muscles, in the regenerating muscles following bupivacaine injection and in the denervated muscle after section of sciatic nerve. Slight TGF-beta 2 immunoreactivity was detected both in slow- and fast-type muscles of mature rat. The amount of TGF-beta RII protein was markedly greater in fast-type muscles. In the overloaded muscle, immunohistochemical analysis showed a marked increase in TGF-beta 2 immunoreactivity in the mononuclear cells (probably endothelial and perithelial or smooth muscle cells of endomysial capillaries) of the extracellular space at 3 and 6 days post surgery. Rapid increase of TGF-beta 2 protein and concomitant decrease of the receptor (TGF-beta RII) were observed in the mechanically overloaded and regenerating muscles. On the other hand, denervation of slow- and fast-type muscles showed a rapid increase in TGF-beta 2 protein, but did not elicit a concomitant decrease of TGF-beta RII. These results indicate that TGF-beta RII is preferentially distributed in fast-type muscles. Furthermore, TGF-beta 2 may play an important role in muscle hypertrophy and regeneration by the usage of TGF-beta RII.

The survival of motoneurons is dependent on them receiving continual trophic support from muscle fibres and various other cell types. Numerous putative survival factors have been identified and a set of criteria established by which these candidates can be assessed. These criteria include the need for the factor and its receptors to be in appropriate locations and for the factor or its second message to be retrogradely transported. In this paper, we demonstrate that a multifunctional cytokine, transforming growth factor-beta 2, appears to meet these criteria. The locations of the transforming growth factor-beta 2 and its receptors in the neuromuscular system were determined by reverse transcriptase-polymerase chain reaction and immunohistochemistry. Motoneurons were shown to synthesize the three proteins involved in transforming growth factor-beta 2 signalling (types I and II transforming growth factor-beta receptor and betaglycan) and to transport them anterogradely, where they were inserted into the axonal membrane and nerve terminal. Transforming growth factor-beta 2 was detected in the synaptic portions of muscle fibres, motoneurons and in injured nerves, indicating that motoneurons may be exposed to multiple and potentially redundant sources of transforming growth factor-beta 2. Double-ligation experiments were used to demonstrate that motoneurons transport transforming growth factor-beta 2 up and down their axons. The anterograde transport of both transforming growth factor-beta 2 and its receptors, coupled with the fact that most of a motoneuron's mitochondria are located in the axon, raises the issue of whether the repression of the initiation of apoptosis is restricted to the cell body or occurs along the entire length of a neuron.

Expression of transforming growth factor (TGF)-beta(3) as well as cellular localization of TGF-beta receptors has not been demonstrated in placenta. TGF-beta receptor type I (RI) and type II (RII) are required to transmit TGF-beta signals, therefore the determination of cells expressing both receptors in concert is necessary to identify target cells for TGF-beta. We investigated presence and localization of TGF-betas and their receptors (RI, RII) in human term placenta using reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. RT-PCR showed that messenger RNA for TGF-beta(1), -beta(2), -beta(3), and RI and RII is present in human term placenta. We found intense staining for all TGF-beta isoform and receptor proteins in the syncytiotrophoblastic layer, chorionic plate, and in cells of the extravillous trophoblasts using immunohistochemistry. The simultaneous expression of ligands and their receptors support the hypothesis that TGF-beta may play an important role in regulating growth, differentiation, and function of the human placenta., (Copyright 2000 S. Karger AG, Basel)

Unlabelled: Increased expression of TGF-beta1 but not of its receptors contributes to human obstructive nephropathy., Background: Previous studies have revealed an increased expression of transforming growth factor-beta1 (TGF-beta1) and deposition of extracellular matrix in the kidney of animals with ureteral obstruction. However, these relationships have not been elucidated in the hydronephrotic kidney of humans., Methods: We analyzed the tissue expression of extracellular matrix proteins, TGF-beta1, and its receptors in the human kidney with ureteral obstruction by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). Obstructed kidneys (OBKs) were obtained from patients with ureteral tumors. A kidney specimen from patients with a renal tumor was used as control (CNKs)., Results: The interstitial volume was significantly increased in OBKs in comparison with CNKs. OBKs showed increased deposition of collagen types I and IV and fibronectin in the renal interstitium. RT-PCR revealed overexpression of collagen alpha1(IV) mRNA and fibronectin mRNA in OBKs. OBKs showed a significantly increased mRNA expression of TGF-beta1 in comparison with CNKs. The immunoreactivity for TGF-beta1 increased markedly in the interstitium of OBKs. There was a significant correlation between the TGF-beta1 mRNA level and the interstitial volume. However, there was no significant difference between OBKs and CNKs in the relative mRNA level nor in immunoreactivity for TGF-beta receptors., Conclusions: These data suggest that TGF-beta1 may contribute to the interstitial fibrosis found in the human kidney with ureteral obstruction, mainly because of an increase in the expression of this cytokine without significant changes to its receptors.

Mesenchymal stem cells are multipotent cells resident in the bone marrow throughout adulthood which have the capacity to differentiate into cartilage, bone, fat, muscle, and tendon. A number of monoclonal antibodies raised against human MSCs have been shown to react with surface antigens on these cells in vitro. A protein of molecular mass 92 kDa was immunoprecipitated using the SH-2 monoclonal antibody. This was purified and identified by peptide sequencing analysis and mass spectrometry as endoglin (CD105), the TGF-beta receptor III present on endothelial cells, syncytiotrophoblasts, macrophages, and connective tissue stromal cells. Endoglin on MSCs potentially plays a role in TGF-beta signalling in the control of chondrogenic differentiation of MSCs and also in mediating interactions between MSCs and haematopoietic cells in the bone marrow microenvironment., (Copyright 1999 Academic Press.)

Freshly isolated human mucosal T lymphocytes in vitro can markedly diminish an important property of intestinal epithelium-its barrier function. On the other hand, cytokines and their cellular receptors, which maintain homeostasis of epithelia, limit epithelial permeability, and preserve barrier function, are not well characterized. Using a described human colonic epithelial cell monolayer system, we found that transforming growth factor-beta1 (TGF-beta1) preserved 75% or more of epithelial barrier function, quantitated electrophysiologically, even in the presence of cytokines generated by a high density of barrier-disruptive mucosa-derived mononuclear cells. In opposing the TGF-beta1 effect, cytokines able to reduce barrier function were spontaneously secreted by mucosal T cells and were increased in their barrier effect after T-lymphocyte activation. Further, neutralization of individual cytokines with specific monoclonal antibodies abrogated the lymphocyte-induced reduction in epithelial barrier function, and identified interferon gamma (IFN-gamma), interleukin (IL)-4, and IL-10, but not IL-6, as the primary cytokines whose barrier effects were curtailed by TGF-beta1. Receptors (RI and RII) for TGF-beta1 were found to be localized primarily to the apical and basal membranes of surface epithelium in colonic crypts. These findings provide the scientific basis for new strategies to pharmacologically enhance the barrier function of epithelia in mucosal organs regularly exposed to environmental antigens and to T-lymphocyte products., (Copyright 1999 Wiley-Liss, Inc.)

Numerous spontaneous and experimentally induced mouse mutations develop a hair phenotype, which is often associated with more or less discrete abnormalities in hair follicle development. In order to recognize these, it is critically important to be able to determine and to classify accurately the major stages of normal murine hair follicle morphogenesis. As an aid, we propose a pragmatic and comprehensive guide, modified after previous suggestions by Hardy, and provide a list of easily recognizable classification criteria, illustrated by representative micrographs. Basic and more advanced criteria are distinguished, the former being applicable to all mouse strains and requiring only simple histologic stains (hematoxylin and eosin, Giemsa, periodic acid Schiff, alkaline phosphatase activity), the latter serving as auxiliary criteria, which require a pigmented mouse strain (like C57BL/6J) or immunohistochemistry (interleukin-1 receptor type I, transforming growth factor-beta receptor type II). In addition, we present simplified, computer-generated schematic drawings for the standardized recording and reporting of gene and antigen expression patterns during hair follicle development. This classification aid serves as a basic introduction into the field of hair follicle morphogenesis, aims at standardizing the presentation of related hair research data, and should become a useful tool when screening new mouse mutants for discrete abnormalities of hair follicle morphogenesis (compared with the respective wild type) in a highly reproducible, easily applicable, and quantifiable manner.

Transforming growth factor (TGF)-beta regulates cell growth and differentiation, and is known to play regulatory roles in the process of tissue repair and remodeling. However, the functional role of TGF-beta in gastric ulcer healing has not been addressed. In this study, we assayed the expression of TGF-beta and its receptors in the gastric mucosa of patients with healed or refractory gastric ulcers. Antibodies against TGF-beta and its receptors (both type I and type II) were employed to examine expression levels. Sixteen gastric ulcer patients, including four with completely healed ulcers and 12 with ulcers refractory to treatment were included in this study. All four patients with healed ulcers showed remarkable expression levels of both TGF-beta and its receptors. On the other hand, two of the 12 patients with refractory ulcers had weak or deficient TGF-beta expression in the gastric mucosa, and seven lacked expression of at least one of the TGF-beta receptors. The remaining three patients had normal (moderate to weak) expression levels of TGF-beta and its two receptors. These results suggest that both TGF-beta and its receptors are essential for gastric ulcer healing.

Objective: Comparative analysis of transforming growth factor-beta 1 (TGF-beta 1) and TGF-beta receptor type I and type II messenger RNA (mRNA) and protein expression in myometrium of women who had unsuccessful labor induction, with those without labor or in preterm labor complicated by chorioamnionitis., Methods: Small segments of myometrium were collected from women who were undergoing cesarean delivery for unsuccessful labor induction (n = 5), elective cesarean without labor (n = 5), or cesarean delivery for complications related to preterm labor and chorioamnionitis (n = 5). Total RNA was isolated from these tissues and subjected to competitive quantitative reverse-transcription-polymerase chain reaction (Q-RT-PCR) to determine the level of TGF-beta 1, and TGF-beta type I and type II receptor mRNA expression. Tissue sections were prepared from paraffin-embedded specimens and immunostained for TGF-beta 1 and receptor proteins using specific polycolonal antibodies. The data were analyzed by impaired Student t test and Kruskal-Wallis analysis of variance., Results: Myometrium from women who had unsuccessful labor induction expressed higher levels of TGF-beta 1 mRNA (2.21 +/- 0.28 x 10(6) copies/microgram of total cellular RNA) than those with preterm labor (4.53 +/- 0.2 x 10(5) copies), or without labor [3.13 +/- 2.6 x 10(4) copies (P < .05)]. The level of TGF-beta type I receptor mRNA expression did not vary; however, type II receptor expression was significantly lower in myometrium from preterm labor (1.36 +/- 0.36 x 10(5) copies) compared with those from unsuccessful labor induction (3.42 +/- 0.42 x 10(6) copies) or without labor (9.65 +/- 3.2 x 10(5) copies). Immunoreactive TGF-beta 1 and TGF-beta receptor proteins were present in all myometrial tissues, and their intensity reflected that of the mRNA expression in these tissues., Conclusion: TGF-beta 1 and TGF-beta type II receptors are expressed differently in myometrium of women who had unsuccessful labor induction compared with those without labor or with preterm labor complicated by chorioamnionitis. Because TGF-beta is a key regulator of tissue remodeling which is central to initiation of normal labor, alterations in TGF-beta and/or TGF-beta receptor expression may lead to changes in the outcome of labor, at least at the myometrial level.

Transforming growth factor-betas (TGF-beta s) and bone morphogenetic proteins (BMPs), members of a TGF-beta superfamily, are known to play an important role in osteogenic cell differentiation and consequently bone formation. We have reported previously that periodontal ligament (PDL) cells differentiate and form mineralized nodules when cultured in the presence of dexamethasone (Dex), beta-glycerophosphate (GP) and ascorbic acid (AA). To understand the roles of TGF-beta isoforms (TGF-beta 1, 2 and 3) and TGF-beta type I receptors (activin receptor-like kinase (ALK)-2, -3, -5 and -6) in PDL cell differentiation, their expression was investigated using Northern blot analysis. Rat PDL cells, derived from coagulum in the tooth socket, were cultured in the presence of Dex (5 microM), GP (10 mM) and AA (50 micrograms/ml) for up to 21 d. Total RNA was isolated from PDL cells after 0, 7, 14 and 21 d and used for northern blot analysis of mRNAs for matrix proteins, TGF-beta isoforms and their receptors using 32P-labeled cDNAs as probes. Four stages showing distinct morphological characteristics and matrix expression during development of mineralized nodules were identified. Type I collagen (Col I) and SPARC (secreted protein, acidic and rich in cysteine) mRNAs were expressed at the confluent stage, but decreased during the mineralization stage. Osteopontin (OPN) and alkaline phosphatase (ALP) transcripts were initially observed at multilayer stage, while bone sialoprotein (BSP) and osteocalcin (OC) at the nodule stage and all 4 were expressed thereafter. TGF-beta 1 mRNA expression increased with the progression of PDL cell differentiation, while a relatively high level of TGF-beta 3 transcript decreased slightly during their differentiation. TGF-beta 2 mRNA was not expressed. The expression of TGF beta-RI mRNA decreased, whereas that of TGF beta-RIII increased dramatically with PDL cell differentiation. TGF beta-RII gene activities remained high throughout all stages. ALK-2, ALK-3 and ALK-6 mRNA expression increased with the progression of PDL cell differentiation, suggesting that these receptors may play important roles in Dex-induced PDL cell differentiation and mineralized nodule formation.

Background: Transforming growth factor-beta (TGF-beta) is a causal factor in experimental glomerulosclerosis, and it mediates the increased extracellular matrix (ECM) accumulation that occurs in cultured mesangial cells (MCs) exposed to high glucose concentrations and cyclic mechanical strain. This change is associated with increased levels of TGF-beta, but may also involve alterations in receptor expression and binding., Methods: Rat MCs cultured in media containing either 8 or 35 mM glucose were seeded into culture plates with elastin-coated flexible bottoms. Thereafter, they were subjected to cyclic stretch or static conditions and then examined for 125I-TGF-beta1 binding and expression of TGF-beta receptors at the gene and protein levels., Results: Kinetic studies showed that MCs bound TGF-beta1 in a time- and concentration-dependent manner, expressing 6800 high-affinity receptors per cell, with an apparent dissociation constant (Kd) of 15.4 pM, while cross-linking analysis identified three TGF-beta receptors (betaR) corresponding to betaRI, betaRII, and betaRIII of 54, 73, and 200 kDa, respectively. Immunocytochemical studies of betaRI and betaRII protein revealed MC expression in a homogeneous, punctate distribution, whereas Northern analysis demonstrated the presence of the corresponding mRNAs. Exposure to cyclic stretching significantly increased (10%) the overall number of TGF-beta receptors, whereas ligands associated with betaRs I, II, and III also increased (25 to 50%). The finding of increased (30 to 40%) betaRI and betaRII transcript levels and immunoreactive protein (163 and 59%, respectively) in the absence of significant changes in the apparent Kd indicated that stretch-induced binding was the result of increased receptor synthesis and expression and not due to a change in binding affinity. In a similar, but more dramatic fashion, exposure to high glucose also elevated (50%) the receptor number, as well as the amount of ligands associated with betaRs I, II, and III (100 to 250%). This same treatment also increased the levels of betaRI and betaRII mRNA (30 to 40%) and the immunoreactive protein (82 and 82%, respectively), without significantly altering the binding affinity of the receptor. A concerted or synergistic effect of both stimuli was not evidenced., Conclusion: These results suggest that the modulation of TGF-beta receptors may be an additional control point in mediating the glucose- and mechanical force-induced increase in ECM deposition by MCs.

Pulmonary hypoplasia has been found in the human neonatal autopsy population and has been attributed to an alteration in epithelial-mesenchymal interactions during development of the lung. Pulmonary acinar aplasia is a very rare and severe form of pulmonary hypoplasia. The transforming growth factor-betas (TGF-beta) are multifunctional regulatory peptides that are secreted by a variety of normal and malignant cells and are expressed in developing organs including the lung; their tissue distribution patterns have possible significance for signaling roles in many epithelial-mesenchymal interactions. Here, we report our examination of TGF-beta in the lungs of a term female infant diagnosed with pulmonary acinar aplasia whose autopsy revealed extremely hypoplastic lungs with complete absence of alveolar ducts and alveoli. Immunohistochemical and in situ hybridization analyses were used to localize and measure the proteins and mRNA, respectively, for TGF-beta1, TGF-beta2, TGF-beta3, and TGF-beta type I and type II receptors (TGF-beta RI and RII) in formalin-fixed and paraffin-embedded sections of these hypoplastic lungs and normal lungs. Immunostaining for TGF-beta1, TGF-beta2, and TGF-beta RI and RII was significantly lower in the bronchial epithelium and muscle of the hypoplastic lungs than in normal lungs, whereas no difference was detected in staining for other proteins including Clara cell 10-kD protein, adrenomedullin, hepatocyte growth factor/scatter factor, and hepatocyte growth factor receptor/Met in the hypoplastic and normal lungs or in the liver and kidneys of this infant compared with normal liver and kidney. In addition, in situ hybridization showed that TGF-beta1 and TGF-beta RI transcripts were considerably reduced in the bronchial epithelium of the hypoplastic lung compared with normal lung. These results show that there is a selective reduction of TGF-beta in pulmonary acinar aplasia and suggest that the signaling action of TGF-beta in epithelial-mesenchymal interactions in the lungs of this developmental condition may be compromised.

Objective: To evaluate the parameters that mediate fibrogenesis in chronic pancreatitis (CP)., Background: Connective tissue growth factor (CTGF), which is regulated by transforming growth factor beta (TGF-beta), has recently been implicated in skin fibrosis and atherosclerosis. In the present study, the authors analyzed the concomitant presence of TGF-beta1 and its signaling receptors-TGF-beta receptor I, subtype ALK5 (TbetaR-I(ALK5)), and TGF-beta receptor II (TbetaR-II)-as well as CTGF and collagen type I in the pancreatic tissue of patients undergoing surgery for chronic pancreatitis., Patients and Methods: CP tissue samples were obtained from 40 patients (8 women, 32 men) undergoing pancreatic resection. Tissue samples of 25 previously healthy organ donors (12 women, 13 men) served as controls. The expression of TGF-beta1, TbetaR-I(ALK5), TbetaR-II, CTGF, and collagen type I was studied by Northern blot analysis. By in situ hybridization and immunohistochemistry, the respective mRNA moieties and proteins were localized in the tissue samples., Results: Northern blot analysis showed that CP tissue samples exhibited concomitant enhanced mRNA expression of TGF-beta1 (38-fold), TbetaR-II (5-fold), CTGF (25-fold), and collagen type I (24-fold) compared with normal controls. In addition, TbetaR-I(ALK5) mRNA was increased in 50% of CP tissue samples (1.8-fold). By in situ hybridization, TGF-beta1, TbetaR-I(ALK5), and TbetaR-II mRNA were often seen to be colocalized, especially in the ductal cells and in metaplastic areas where atrophic acinar cells appeared to dedifferentiate into ductal structures. In contrast, CTGF was located in degenerating acinar cells and principally in fibroblasts surrounding these areas. Moreover, CTGF mRNA expression levels correlated positively with the degree of fibrosis in CP tissues., Conclusion: The concomitant overexpression of CTGF, collagen type I, TGF-beta1, and its signaling receptors in CP suggests that these proteins contribute to enhanced extracellular matrix synthesis and accumulation, resulting finally in the fibrogenesis observed in CP.

Circumstantial evidence from clinical and pathologic correlations in patients with glomerular diseases and proteinuria suggest that glomerular protein ultrafiltration contributes to tubulointerstitial injury. A series of studies was performed to examine the hypothesis that in rats with adriamycin-induced nephropathy or with diabetic nephropathy (but not in normal rats) high molecular wt. growth factors are ultrafiltered into tubular fluid and act on tubular cells through apical membrane receptors. Analysis of proximal tubular fluid that was collected by nephron micropuncture indicates ultrafiltration of IGF-I, TGF-beta and HGF. Respective receptors are also expressed in apical membranes in some parts of the nephron as examined by immunohistochemistry. In vitro cell culture experiments using proximal tubular fluid obtained from rats with experimental glomerular diseases indicate that ultrafiltered IGF-I may contribute to increased distal tubular Na-absorption. Indirect evidence also suggests that this growth factor may increase the secretion of collagen types I and IV in proximal tubular cells. TGF-beta and HGF cause increased expression and basolateral secretion of MCP-1 in proximal tubular and collecting duct cells. There may be other biologic effects on tubules that are caused by apical exposure to ultrafiltered growth factors. These studies suggest that the glomerular ultrafiltration of bioactive proteins causes or contributes to tubulo-interstitial pathology in glomerular proteinuria.

Little is known about the mechanisms controlling the expression of key proteins that regulate excitability and contractility in the human myometrium at term. However, evidence is accumulating to suggest that the cytokine transforming growth factor (TGF)beta may play a central role. TGFbeta1 and TGFbeta receptors are present in the myometrial cells, indicative of an autocrine signaling system. Furthermore, the levels of TGFbeta1 and the expression of its receptors increase in the myometrium at term suggesting that they are, in turn, regulated and form part of a physiological cascade of events involving a number of autocrine signaling associated proteins. The present experiments were done to identify factors that regulate the expression of TGFbeta1 and TGFbeta receptors and may form other elements of this cascade. Because IL-1 and IL-8 are found in the myometrium at term and have been implicated in the etiology in premature labor we focus on this cytokines. Receptors for IL-1 and IL-8 were detected in the myometrial cells. Using Western blot analysis, the levels of expression were found to vary. The expression of IL-1 receptor type I was highest in the nonpregnant tissue with lower levels in nonlaboring myometrium with a further reduction in the spontaneously laboring tissue. In contrast, the expression of IL-8 receptor type B was highest in the pregnant nonlaboring tissue with a lower level in the spontaneously laboring tissue. Using an in vitro model, TGFbeta1 and TGFbeta receptor expression was up-regulated by IL-8, IL-1, and TGFbeta1 itself. However, IL-8 receptor expression was decreased by IL-8 and TGFbeta1. This suggests that in a cascade IL-8 would feed forward to promote the TGFbeta system, whereas TGFbeta1 feeds back to inhibit responsiveness to IL-8. Estrogen and progesterone increased the release of TGFbeta1. However, at high concentrations, estrogen and progesterone (100 nM 17beta-estradiol or 200 nM progesterone) decreased the level of TGFbeta receptor expression. Thus, the progressive rise of steroid levels in vivo might account for the observed changes in TGFbeta1 and TGFbeta receptor expression in vivo. Taken together, these observations support the idea that there is a cascade of autocrine signals that may play a major role in the physiological processes preparing the myometrium for parturition at term.

Exposure of male rats to estrogens during the neonatal period retards prostate branching morphogenesis, blocks epithelial differentiation, and predisposes the adult prostate to hyperplasia and dysplasia. The mechanism of neonatal estrogenization is not well understood. The present study evaluated transforming growth factor-beta (TGFbeta) in the neonatally estrogenized ventral prostate to determine whether this paracrine/autocrine factor may in part mediate the effects ofestrogen on the developing prostate gland. Immunocytochemistry using antibodies against active TGFbeta1 and its latency-associated peptide localized this molecule to the periductal smooth muscle cells in the developing prostate. Although neonatal estrogenization increased the accumulation of total and active TGFbeta1 in the smooth muscle layer as early as day 6 of life, it was physically separated from the epithelial ducts by a proliferating layer of fibroblasts surrounding the basement membrane. RT-PCR demonstrated that alterations in TGFbeta1 levels were not due to alterations in TGFbeta1 transcription. TGFbeta2 and TGFbeta3 were primarily immunolocalized to differentiating epithelial cells in developing prostates, and this was markedly dampened between days 10-30 after neonatal estrogen exposure. Immunocytochemistry for TGFbeta signaling components revealed that neonatal estrogenization transiently reduced TGFbeta type I receptor levels in the prostate epithelium, but not in stroma, between days 6-15, whereas there was no effect on TGFbeta type II receptor. Levels of the intracellular signal Smad2 (52 kDa) were detected in epithelial cells but were not altered after estrogenization. To analyze the functional status of the TGFbeta signaling pathway, immunocytochemistry was performed for p21(cip-1/waf-1), a cyclin-dependent kinase inhibitor that is inducible by TGFbeta1 in the prostate. Transient nuclear localization of p21(cip-1/waf-1) was normally observed in epithelial cells between days 6-15 and was associated with entry of cells into a terminal differentiation pathway. Neonatal estrogenization prevented this transient expression of p21(cip-1/waf-1). The present findings demonstrate that the TGFbeta signaling system is perturbed at several levels in the estrogenized prostate, which may in part account for the epithelial cell differentiation blockade as well as the proliferation of periductal fibroblasts in this model.

Transforming growth factor beta (TGF-beta) is involved in the modulation of cell growth, differentiation and repair following injury of various organs. Previous studies on human autopsy material have indicated that TGF-beta isoforms-beta1, -beta2 and -beta3, and TGF-beta receptor type I are expressed in various cells of necrotizing brain lesions like infarction and abscess. The present immunohistochemical study was designed to investigate changes that may occur with regard to TGF-beta and its receptors type I and II in a rat model of focal brain ischemia induced by transient or permanent occlusion of the middle cerebral artery. Our findings indicate that at days 1 and 3 following such transient and permanent ischemia there is an up-regulation of TGF-beta isoforms -beta1, -beta2 and -beta3, and TGF-beta receptor types I and II mainly in the perifocal neurons, reactive astroglial cells, endothelial cells and macrophages.

Objective: To study the expression of T beta R I in HCC tissues and its clinical significance., Methods: Expressions of T beta R I in HCC tissues (HT, n = 30) and surrounding liver tissues(HST, n = 30) as well as four normal control liver tissues (CIT, n = 4) were determined by radioligand binding assay with 125I TGF beta 1 as the ligand. Relationships between some clinicopathological parameters of HCC and the relative expression rates of T beta R I in HCC were studied., Results: Compared with that in HST, the expression of T beta R I HT was significantly lower than in HST and CIT (P < 0.01). The expression of T beta R I in HST is similar to that in CIT. T beta R I relative expression rates in the group of no capsule or capsule invaded and in the group of tumor embolus were markedly decreased (P < 0.05). No close relationships were found between the T beta R I relative expression rate and age, sex, HBV infection, liver cirrhosis, AFP concentration, tumor diameter, tumor number, tumor differentiation as well as tumor embolus., Conclusions: The low expression of T beta R I in HT may be one of the key event which promote the HCC malignant growth by protecting them against the growth inhibition effect of active TGF beta. The T beta R I relative expression rate may be related to the prognosis of HCC.

It is known that the pleiotropic cytokine transforming growth factor beta (TGF-beta) has a regulatory role in the process of tissue repair and remodelling following injury. As reports on these molecules in multiple sclerosis (MS) lesion with different lesional activity are rare, we studied the cellular localization of TGF-beta1, -beta2, and -beta3 isoforms, and TGF-beta receptor type I (TGF-betaR-I) and TGF-betaR-II expression by immunohistochemistry on postmortem brain tissue from MS and normal control cases. To validate the TGF-beta staining results we demonstrated that cultured human adult astrocytes that produce biological active TGF-beta2, and to a lesser extent TGF-beta1, were immunoreactive for all 3 TGF-beta isoforms. Moreover, at mRNA level TGF-beta1 was detected in MS and normal control brain tissue. In normal control brain tissue, TGF-beta isoforms were expressed in ramified microglia and TGF-beta2, and -beta3 on neuronal cells in the gray matter TGF-betaR-I and TGF-betaR-II expression was found on endothelial cells, astrocytes, microglia, and neurons. In active demyelinating MS lesions a strong to intense immunoreactivity was detected for all 3 TGF-beta isoforms in perivascular and parenchymal (foamy) macrophages and in hypertrophic astrocytes. Strong immunoreactivity for TGF-betaR-I and TGF-betaR-II was found on macrophages in both parenchymal and perivascular areas and on hypertrophic astrocytes and endothelial cells in active demyelinating MS lesions. In chronic active and inactive MS lesions, all 3 TGF-beta isoforms and their receptors were strongly expressed in hypertrophic astrocytes. Our findings strongly suggest that the expression of the various TGF-beta isoforms and their receptor types found in MS lesions with different cellular activity participate in reactive processes leading to the formation of chronic MS lesions.

Objective: To evaluate mechanisms that contribute to tissue repair and tissue remodeling in Crohn's disease (CD)., Summary Background Data: Transforming growth factor-betas (TGF-betas) are involved in different chronic inflammatory disorders. They function by binding to two receptors, type I (TbetaR-I) subtype ALK5 and type II (TbetaR-II), which are concomitantly required for signal transduction., Methods: Tissues were obtained from 18 patients with CD (10 female patients, 8 male patients, median age 38.7 years [range 16 to 58 years]) undergoing surgery because of CD-related complications. Tissue samples of 18 healthy organ donors (10 female subjects, 8 male subjects, median age 50.3 years [range 15 to 65 years]) served as controls. The expression and localization of TGF-beta1, TGF-beta2, TGF-beta3, TbetaR-IALK5, TbetaR-II, and TbetaR-III were studied by Northern blot analysis, in situ hybridization, and immunohistochemistry., Results: On Northern blot analysis, 94% of the CD samples exhibited enhanced TGF-beta1, TGF-beta3, and TbetaR-II mRNA expression compared with controls. TGF-beta2 was increased in 72%, TbetaR-IALK5 in 72%, and TbetaR-III in 82% of the patients with CD. On in situ hybridization and immunohistochemical analysis, TGF-beta1, TbetaR-IALK5, and TbetaR-II were seen to be colocalized in the lamina propria cells and in the lymphocytes closest to the luminal surface, but also in the remaining epithelial cells, and in fibroblasts of CD tissue samples., Conclusions: The concomitant overexpression of TGF-betas and their signaling receptors in CD points to a potential role of these regulatory molecules in the pathophysiology of CD. Activation of TGF-beta-mediated pathways might promote the repair of mucosal injury by enhancing the process of reepithelization, but might also contribute to extracellular matrix generation and subsequently to intramural fibrosis and intestinal obstruction.

Objective: To investigate the role of cell cycle regulators in the pathogenesis of papillary carcinoma of the thyroid., Design: Resistance to transforming growth factor beta-mediated inhibition is a well-known pathogenic mechanism in epithelial neoplasias. In a retrospective study, the expression of transforming growth factor beta receptors types I and II, cyclin D1, and the cyclin-dependent inhibitor p27kip, was analyzed by immunohistochemistry. Results were interpreted in the context of clinicopathological data. Patient follow-up ranged from 1 to 18 years, with a mean of 4 years., Materials: Twenty conventional primary papillary carcinomas and their metastases were selected according to current pathologic criteria. Nonconventional papillary carcinomas (eg, tall-cell, columnar) were excluded from the analysis., Results: Cyclin D1 was expressed more intensely in the tumor than in adjacent nonneoplastic parenchyma. Within a given tumor, however, there was significant heterogeneity in expression intensity and percentage of positive cells, particularly in metastases. Type I receptors were strongly expressed in 90% of tumors, while 80% of the tumors revealed low to no expression of type II receptors. In 10% of tumors, type I receptors were absent and type II receptors expressed. Simultaneous absence of both receptors was not observed. While p27kip was strongly expressed in nonneoplastic thyroid, it was not detected in any of the primary tumors or their metastases., Conclusions: The results strongly suggest that functional abnormalities in type II receptors result in increased levels of cyclin D1 and down-regulation of p27kip. This would maintain cells in a proliferative state and would promote tumor progression.