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Surgically repaired tendons are plagued by complications related to the healing response. Adhesion formation between the tendon and its sheath or surrounding tissues inhibits free gliding and results in a loss of excursion. The random orientation of collagen deposition at the site of repair creates a focal area of weakness, thereby rendering the tendon prone to rupture at this point. Even the strongest, most technically precise repair can be negated by excessive scar tissue. No widely accepted therapy currently exists to promote healing and prevent fibrosis in surgically repaired tendons. Transforming growth factor beta (TGF-β) is considered the active factor during healing that leads to scar formation. It does so by binding with a mannose-6-phosphate/IGF-II receptor on the Golgi apparatus, which changes the extracellular matrix and ultimately leads to fibrosis. Therefore, inhibiting TGF-β may be one method to reduce scar formation. The goal of this study was to determine the effects of mannose-6-phophate (M6P) in inhibiting transforming growth factor β1 (TGF-β1) proliferation of fibroblast cells. McCoy fibroblasts were treated with low, medium, and high concentrations of mannose-6-phosphate for periods of 24, 48, and 72 hours, and with low, medium, and high concentrations of TGF-pi for periods of 24, 48, and 72 hours. Cell proliferation, damage, and morphology were evaluated at each time point. The results show that low dose TGF-Pi treatment resulted in significant increases in cell number with distinct cytological changes within 48 hours of treatment. Mannose-6phosphate reduced cell number within the first 48 hours and appeared to be dose dependent. A competitive assay was then developed using low concentration TGF-β1 and medium concentration M6P at 48 hours to determine if M6P could interfere with TGF-β1-induced fibroblast cell growth. Cell proliferation, damage, and morphology were evaluated. The results show that M6P is capable of reducing TGF-β1-induced fibroblast proliferation, and it is suggested that this effect is through competitive inhibition of the M6P/IGF-II receptor of fibroblasts. Keywords: mannose-6-phosphate, transforming growth factor-β, fibrosis, INTRODUCTION Tendon healing is plagued by the complications of rupture and adhesion formation. Both of these tend to occur early in healing and are due to improper regulation of collagen [...]

In bacteria, the rate of cell proliferation and the level of gene expression are intimately intertwined. Elucidating these relations is important both for understanding the physiological functions of endogenous genetic circuits and for designing robust synthetic systems. We describe a phenomenological study that reveals intrinsic constraints governing the allocation of resources toward protein synthesis and other aspects of ceil growth. A theory incorporating these constraints can accurately predict how ceil proliferation and gene expression affect one another, quantitatively accounting for the effect of translation-inhibiting antibiotics on gene expression and the effect of gratuitous protein expression on cell growth. The use of such empirical relations, analogous to phenomenological laws, may facilitate our understanding and manipulation of complex biological systems before underlying regulatory circuits are elucidated. 10.1126/science.1192588

Various uremic toxins accumulate in patients with chronic renal failure (CRF) and one of them is indoxyl sulfate, which accelerates the progression of CRF through unknown mechanisms. The present study investigates how indoxyl sulfate promotes CRF using the proximal tubular cell line HK-2 and CRF rats. Indoxyl sulfate inhibited serum-induced cell proliferation and promoted the activation of senescence-associated [beta]-galactosidase, a marker of cellular senescence, and the expression of [alpha]-smooth muscle actin ([alpha]-SMA), a marker of fibrosis, through inducing p53 expression and phosphorylation. Pifithrin-[alpha], p-nitro, a p53 inhibitor, blocked these effects. Indoxyl sulfate evoked reactive oxygen species (ROS), and the antioxidant N-acetylcysteine inhibited indoxyl sulfate-induced p53 expression and phosphorylation, as well as indoxyl sulfate-induced [alpha]-SMA expression. We previously demonstrated that although cellular senescence and fibrosis are detectable in the kidneys of CRF rats, the oral adsorbent AST-120 repressed these effects. Here, we found that [beta]-galactosidase, p53 and [alpha]-SMA were expressed and colocalized in the renal tubules of CRF rats, whereas AST-120 decreased the expression of these genes. Taken together, these findings indicate that indoxyl sulfate induces the expression and phosphorylation of p53 though ROS production, thus inhibiting cell proliferation and promoting cellular senescence and renal fibrosis. renal failure; uremic toxin; fibrosis; nephrotoxicity doi: 10.1152/ajpcell.00217.2010.

Identification of renal cell progenitors and recognition of the events contributing to cell regeneration following ischemia-reperfusion injury (IRI) are a major challenge. In a mouse model of unilateral renal IRI, we demonstrated that the first cells to proliferate within injured kidneys were urothelial cells expressing the progenitor cell marker cytokeratin 14. A systematic cutting of the injured kidney revealed that these urothelial cells were located in the deep cortex at the corticomedullary junction in the vicinity of lobar vessels. Contrary to multilayered bladder urothelium, these intrarenal urothelial cells located in the upper part of the medulla constitute a monolayered barrier and express among uroplakins only uroplakin III. However, like bladder progenitors, intrarenal urothelial cells proliferated through a FGF receptor-2 (FGFR2)-mediated process. They strongly expressed FGFR2 and proliferated in vivo after recombinant FGF7 administration to control mice. In addition, IRI led to FGFR phosphorylation together with the selective upregulation of FGF7 and FGF2. Conversely, by day 2 following IRI, renal urothelial cell proliferation was significantly inhibited by FGFR2 antisense oligonucleotide administration into an intrarenal urinary space. Of notice, no significant migration of these early dividing urothelial cells was detected in the cortex within 7 days following IRI. Thus our data show that following IRI, proliferation of urothelial cells is mediated by the FGFR2 pathway and precedes tubular cell proliferation, indicating a particular sensitivity of this structure to changes caused by the ischemic process. FGFR2; ischemia doi: 10.1152/ajprenal.00585.2009.

We previously reported that p38 mitogen-activated protein kinase (p38) and phosphorylated ERK are upregulated in cyst epithelium of human renal dysplasia and obstructive uropathy in fetal lambs (Omori S, Fukuzawa R, Hida M, Awazu M. Kidney Int 61: 899-906, 2002; Omori S, Kitagawa H, Koike J, Fujita H, Hida M, Pringle KC, Awazu M. Kidney Int 73: 1031-1037, 2008). Dysplastic epithelium is characterized by proliferation, apoptosis, and upregulation of Pax2 and transforming growth factor (TGF)-[beta]1. In the present study, we investigated whether cyclic mechanical stretching of ureteric bud cells, a mimic of the hydrodynamic derangement after fetal urinary tract obstruction, reproduces events seen in vivo. Cyclic stretch activated p38 and ERK and upregulated Pax2 expression in a time-dependent manner in ureteric bud cells. Stretch-stimulated Pax2 expression was suppressed by a p38 inhibitor, SB203580, or a MEK inhibitor, PD98059.5-Deoxyuridine incorporation was increased by stretch at 24 h, which was also abolished by SB203580 or PD98059. On the other hand, apoptosis was not induced at 24 h by stretch but was significantly increased at 48 h. TGF-[beta]1 secretion was increased by stretch at 24 h, which was inhibited by SB203580 or PD98059. Inhibition of p38 or ERK as well as anti-TGF-[beta] antibody abolished the stretch-induced apoptosis. Finally, exogenous TGF-[beta]1 induced apoptosis of ureteric bud cells, which was inhibited by SB203580 and PD98059. In conclusion, cyclic stretch induces Pax2 upregulation, proliferation, and TGF-[beta]1-mediated apoptosis, features characteristic of dysplastic epithelium, via p38 and ERK in ureteric bud cells. dysplastic kidney; obstructive uropathy; kidney development; cell signaling doi: 10.1152/ajprenal.00402.2009.

Regenerative medicine holds the promise of replacing damaged tissues largely by stem cell activation. Hedgehog signaling through the plasma membrane receptor Smoothened (Smo) is an important process for regulating stem cell proliferation. The development of Hedgehog-related therapies has been impeded by a lack of US Food and Drug Administration (FDA)-approved Smo agonists. Using a high-content screen with cells expressing Smo receptors and a [beta]-arrestin2-GFP reporter, we identified four FDA-approved drugs, halcinonide, fluticasone, clobetasol, and fluocinonide, as Smo agonists that activate Hedgehog signaling. These drugs demonstrated an ability to bind Smo, promote Smo internalization, activate Gli, and stimulate the proliferation of primary neuronal precursor cells alone and synergistically in the presence of Sonic Hedgehog protein. Halcinonide, fluticasone, clobetasol, and fluocinonide provide an unprecedented opportunity to develop unique clinical strategies to treat Hedgehog-dependent illnesses. steroids | Hedgehog signaling | Gli | stem cell proliferation | arrestin doi: 10.1073/pnas.0910712107

Although mammalian hearts show almost no ability to regenerate, there is a growing initiative to determine whether existing cardiomyocytes or progenitor cells can be coaxed into eliciting a regenerative response. In contrast to mammals, several non-mammalian vertebrate species are able to regenerate their hearts (1-3), including the zebrafish (4,5), which can fully regenerate its heart after amputation of up to 20% of the ventricle. To address directly the source of newly formed cardiomyocytes during zebrafish heart regeneration, we first established a genetic strategy to trace the lineage of cardiomyocytes in the adult fish, on the basis of the Cre/lox system widely used in the mouse (6). Here we use this system to show that regenerated heart muscle cells are derived from the proliferation of differentiated cardiomyocytes. Furthermore, we show that proliferating cardiomyocytes undergo limited dedifferentiation characterized by the disassembly of their sarcomeric structure, detachment from one another and the expression of regulators of cell-cycle progression. Specifically, we show that the gene product of polo-like kinase 1 (plk1) is an essential component of cardiomyocyte proliferation during heart regeneration. Our data provide the first direct evidence for the source of proliferating cardiomyocytes during zebrafish heart regeneration and indicate that stem or progenitor cells are not significantly involved in this process., During heart regeneration in zebrafish, lost ventricular tissue is rapidly replaced. After as little as 1 month, most of the missing tissue has been regenerated by cardiomyocytes. The exact source [...]

Akt is involved in the regulation of diverse cellular functions such as cell proliferation, energy metabolism, and apoptosis. Although three Akt isoforms are known, the function of each isoform is poorly understood. To gain a better understanding of each Akt isoform, we examined the subcellular localization and expression of each isoform in transformed and nontransformed cells. Aktl was localized in the cytoplasm, which is in agreement with the currently accepted model that cytoplasmic Akt is translocated and activated at the inner leaflet of the plasma membrane. Interestingly, HEK-293 and HEK-293T cells contained Aktl in the nucleus and cytoplasm, respectively, suggesting that SV40 T-antigen plays a crucial role in the cytoplasmic localization and activation of Aktl in HEK-293T. Akt2 was colocalized with the mitochondria, while Akt3 was localized in both the nucleus and nuclear membrane. The subcellular localization of the Akt isoforms was not substantially altered in response to ionizing radiation or EGF. Furthermore, the ablation of one Akt isoform by small interfering RNA (siRNA) did not alter the subcellular location of the remaining isoforms, suggesting that the major function of one isoform is not compensated for by other isoforms. Together, our data support the notion that Akt2 and Akt3 are regulated at the mitochondrial and nuclear membranes, respectively. The mitochondrial localization of Akt2 raises the possibility that this isoform may be involved in both glucose-based energy metabolism and suppression of apoptosis, two Akt functions previously identified with anti-pan-Akt antibodies. phosphatidylinositol 3-kinase; signal transduction; subcellular localization; mitochondria; radiation doi:10.1152/ajpcell.00375.2009

Shockwaves elicited by transient pressure disturbances are used to treat musculoskeletal disorders. Previous research has shown that shockwave treatment affects T-cell function, enhancing T-cell proliferation and IL-2 expression by activating p38 mitogen-activated protein kinase (MAPK) signaling. Here we investigated the signaling pathway by which shockwaves mediate p38 MAPK phosphorylation. We found that shockwaves at an intensity of 0.18 mJ/m[m.sup.2] induce the release of extracellular ATP from human Jurkat T-cells at least in part by affecting cell viability. ATP released into the extracellular space stimulates P2X7-type purinergic receptors that induce the activation of p38 MAPK and of focal adhesion kinase (FAK) by phosphorylation on residues Tyr397 and Tyr576/577. Elimination of released ATP with apyrase or inhibition of P2X7 receptors with the antagonists KN-62 or suramin significantly weakens FAK phosphorylation, p38 MAPK activation, IL-2 expression, and T-cell proliferation. Conversely, addition of exogenous ATP causes phosphorylation of FAK and p38 MAPK. Silencing of FAK expression also reduces these cell responses to shockwave treatment. We conclude that shockwaves enhance p38 MAPK activation, IL-2 expression, and T-cell proliferation via the release of cellular ATP and feedback mechanisms that involve P2X7 receptor activation and FAK phosphorylation. p38 mitogen-activated protein kinase; ATP; focal adhesion kinase doi:10.1152/ajpcell.00342.2009

Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths worldwide, with a mortality rate approximating its incidence. Understanding the biology of these tumors, as well as treatment modalities, has been challenging. The opioid growth factor (OGF; [[Met.sup.5]]-enkephalin) and the OGF receptor (OGFr) form an endogenous growth-regulating pathway in homeostasis and neoplasia. In this investigation, we examined the relationship of the OGF-OGFr axis in HCC and define its presence, function, and mechanism. Using SK-HEP-1, Hep G2, and Hep 3B human HCC cell lines, we found that OGF and OGFr were present and functional. Exogenous OGF was observed to have a dose-dependent, reversible, and receptor-mediated inhibitory action on cell proliferation. Endogenous OGF was found to be constitutively produced and tonically active on cell replicative activities, with neutralization of this peptide accelerating cell proliferation. Silencing of OGFr using siRNA stimulated cell replication, even when exogenous OGF was added to the cultures, documenting its importance in mediating OGF activity. The mechanism of OGF-OGFr action on cell number was related to inhibition of DNA synthesis and not to apoptotic or necrotic pathways. Both OGF and OGFr were detected in surgical specimens of HCC, and no quantitative differences were recorded in peptide or receptor between pathological and normal specimens. These data are the first to report that the OGF-OGFr system is a native biological regulator of cell proliferation in HCC. The findings may provide important insight in designing treatment strategies for this deadly disease. hepatocellular carcinoma; cell proliferation; tissue culture; siRNA; therapy doi: 10.1152/ajprcgu.00646.2009

Mechanical stress is an important modulator of lung morphogenesis, postnatal lung development, and compensatory lung regrowth. The effect of mechanical stress on stem or progenitor cells is unclear. We examined whether proliferative responses of epithelial progenitor cells, including dually immunoreactive (CCSP and proSP-C) progenitor cells (CCSP+/SP-C+) and type II alveolar epithelial cells (ATII), are affected by physical factors found in the lung of emphysematics, including loss of elastic recoil, reduced elastin content, and alveolar destruction. Mice underwent single lung pneumonectomy (PNY) to modulate transpulmonary pressure (mechanical stress) and to stimulate lung regeneration. Control mice underwent sham thoracotomy. Plombage of different levels was employed to partially or completely abolish this mechanical stress. Responses to graded changes in transpulmonary pressure were assessed in elastin-insufficient mice (elastin +/-, ELN+/-) and elastase-treated mice with elastase-induced emphysema. Physiological regrowth, morphometry (linear mean intercept; Lmi), and the proliferative responses of CCSP+/SP-C+, Clara cells, and ATII were evaluated. Plombage following PNY significantly reduced transpulmonary pressure, regrowth, and CCSP+/SP-C+, Clara cell, and ATII proliferation following PNY. In the ELN+/- group, CCSP+/SP-C+ and ATII proliferation responses were completely abolished, although compensatory lung regrowth was not significantly altered. In contrast, in elastase-injured mice, compensatory lung regrowth was significantly reduced, and ATII but not CCSP+/SP-C+ proliferation responses were impaired. Elastase injury also reduced the baseline abundance of CCSP+/SP-C+, and CCSP+/SP-C+ were found to be displaced from the bronchioalveolar duct junction. These data suggest that qualities of the extracellular matrix including elastin content, mechanical stress, and alveolar integrity strongly influence the regenerative capacity of the lung, and the patterns of cell proliferation in the lungs of adult mice. pneumocyte; bronchioalveolar stem cell; elastase; elastin; pneumonectomy; lung regeneration doi: 10.1152/ajplung.90594.2008.

Control of organ size by cell proliferation and survival is a fundamental developmental process, and its deregulation leads to cancer. However, the molecular mechanism underlying organ size control remains elusive in vertebrates. In Drosophila, the Hippo (Hpo) signaling pathway controls organ size by both restricting cell growth and proliferation and promoting cell death. Here we investigated whether mammals also require the Hpo pathway to control organ size and adult tissue homeostasis. We found that Mst1 and Mst2, the two mouse homologs of the Drosophila Hpo, control the sizes of some, but not all organs, in mice, and Mst1 and Mst2 act as tumor suppressors by restricting cell proliferation and survival. We show that Mst1 and Mst2 play redundant roles, and removal of both resulted in early lethality in mouse embryos. Importantly, tumors developed in the liver with a substantial increase of the stem/progenitor cells by 6 months after removing Mst1 and Mst2 postnatally. We show that Mst1 and Mst2 were required in vivo to control Yap phosphorylation and activity. Interestingly, apoptosis induced by TNF[alpha] was blocked in the Mst1 and Mst2 double-mutant cells both in vivo and in vitro. As TNF[alpha] is a pleiotropic inflammatory cytokine affecting most organs by regulating cell proliferation and cell death, resistance to TNF[alpha]-induced cell death may also contribute significantly to tumor formation in the absence of Mst1 and Mst2. Hippo signaling | tumor suppressor | Yap phosphorylation doi/ 10.1073/pnas.0911409107

Am J Physiol Renal Physiol 298: F209-F215, 2010. First published October 14, 2009; doi: 10.1152/ajprenal.00451.2009.--Episodes of acute renal failure (ARF) are not always fully reversible and may lead to chronic disease, due in part to an inadequate regenerative response. The Notch signaling pathway is involved in determining cell fate during development, and tissue maintenance and repair in adult organs. The purpose of this study was to examine the role of the Notch pathway in renal regeneration following ARF. Kidney injury, induced by ischemia-reperfusion, resulted in early activation of the Notch pathway, as evidenced by increased expression of Notch1 and Notch2 intracellular domain (cleaved Notch). The effect of exogenous administration of the Notch ligand Delta-like-4 (DLL4) on recovery from ARF was then studied. Rats were pretreated by intraperitoneal injection of DLL4 or vehicle control. Two days following the last DLL4 dose, ARF was induced by bilateral renal artery clamping for 45 min followed by reperfusion. The severity of renal injury was similar in DLL4 and control rats. Renal recovery was facilitated by DLL4 treatment, as evidenced by faster return of serum creatinine to baseline by 48 h in DLL4-treated rats as against 5 days in vehicle-treated control rats. Cell proliferation was higher in the DLL4-treated group. In conclusion, activation of the Notch pathway occurs following ARF. Pretreatment with the Notch ligand DLL4 enhanced recovery from ARF and represents a potential novel therapeutic option for regenerating the injured kidney. Delta-like-4; acute kidney injury; cellular proliferation; ischemia-reperfusion

Dmrt1 (doublesex and mab-3 related transcription factor 1) is a conserved transcriptional regulator of male differentiation required for testicular development in vertebrates. Here, we show that in mice of the 129Sv strain, loss of Dmrt1 causes a high incidence of teratomas, whereas these tumors do not form in Dmrt1 mutant C57BL/6J mice. Conditional gene targeting indicates that Dmrt1 is required in fetal germ cells but not in Sertoli cells to prevent teratoma formation. Mutant 129Sv germ cells undergo apparently normal differentiation up to embryonic day 13.5 (E13.5), but some cells fail to arrest mitosis and ectopically express pluripotency markers. Expression analysis and chromatin immunoprecipitation identified DMRT1 target genes, whose missexpression may underlie teratoma formation. DMRT1 indirectly activates the GDNF coreceptor Ret, and it directly represses the pluripotency regulator Sox2. Analysis of human germ cell tumors reveals similar gene expression changes correlated to DMRT1 levels. Dmrt1 behaves genetically as a dose-sensitive tumor suppressor gene in 129Sv mice, and natural variation in Dmrt1 activity can confer teratoma susceptibility. This work reveals a genetic link between testicular dysgenesis, pluripotency regulation, and teratoma susceptibility that is highly sensitive to genetic background and to gene dosage. GDNF | testicular teratoma | germ cell tumor | embryonal carcinoma doi/10.1073/pnas.0905431106

Wang S, Li Y. Expression of constitutively active cGMP-dependent protein kinase inhibits glucose-induced vascular smooth muscle cell proliferation. Am J Physiol Heart Circ Physiol 297: H2075-H2083, 2009. First published August 28, 2009; doi: 10.1152/ajpheart.00521.2009.--Previously, we have demonstrated that cGMP-dependent protein kinase (PKG) activity is downregulated in vessels from diabetic animals or in vascular smooth muscle cells (VSMCs) exposed to high-glucose conditions, contributing to diabetes-associated vessel dysfunction. However, whether decreased PKG activity plays a role in hyperglycemia-induced proliferation of VSMCs is unknown. In this report, high-glucose-mediated decreased PKG activity in VSMCs was restored by transfection of cells with expression vector for the catalytic domain of PKG-I (PKG-CD, constitutive active PKG). The effect of glucose on cell proliferation was determined. Our data demonstrated that high glucose exposure stimulated VSMC proliferation and [G.sub.1] to S phase progression of the cell cycle, which was inhibited by restoration of PKG activity. Expression of constitutively active PKG inhibited [G.sub.1] phase exit in VSMCs under high glucose conditions, which was accompanied by an inhibition of retinoblastoma protein (Rb) phosphorylation (a key switch for [G.sub.1] to S phase cell cycle progression). Glucose-induced cyclin E expression and cyclin E-cyclin-dependent kinase 2 activity was also reduced by expression of PKG-CD in VSMCs. Moreover, expression of PKG-CD suppressed glucose-induced p27 degradation. These data demonstrate that restoring the high-glucose-mediated decrease in PKG activity in VSMCs inhibits glucose-induced abnormal VSMC proliferation occurring upstream of Rb phosphorylation. Our work provides the first direct evidence linking decreased PKG activity to high glucose-induced proliferation and cell cycle progression in VSMCs, suggesting that strategies to increase PKG activity might be useful in preventing abnormal VSMC proliferation in diabetic patients and might provide treatments for diabetes-associated proliferative vascular diseases. guanosine 5'-cyclic monophosphate-dependent protein kinase; vascular smooth muscle cells; cell proliferation; cell cycle; glucose doi: 10.1152/ajpheart.00521.2009

Torella D, Gasparri C, Ellison GM, Curcio A, Leone A, Vicinanza C, Galuppo V, Mendicino I, Sacco W, Aquila I, Surace FC, Luposeila M, Stillo G, Agosti V, Cosentino C, Avvedimento EV, Indolfi C. Differential regulation of vascular smooth muscle and endothelial cell proliferation in vitro and in vivo by cAMP/PKA-activated p85[[alpha].sup.PI3K]. Am J Physiol Heart Circ Physiol 297: H2015-H2025, 2009. First published September 25, 2009; doi: 10.1152/ajpheart.00738.2009.--cAMP inhibits proliferation in most cell types, triggering different and sometimes opposing molecular pathways, p85[alpha] (phosphatidylinositol 3-kinase regulatory subunit) is phosphorylated by cAMP/PKA in certain cell lineages, but its effects on vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) are unknown. In the present study, we evaluated 1) the role of p85[alpha] in the integration of cAMP/PKA-dependent signaling on the regulation of VSMC and EC growth in vitro; and 2) the effects of PKA-modified p85[alpha] on neointimal hyperplasia and endothelial healing after balloon injury in vivo. Plasmid constructs carrying wild-type and PKA-modified p85[alpha] were employed in VSMCs and ECs in vitro and after balloon injury in rat carotid arteries in vivo. cAMP/PKA reduced VSMC proliferation through p85[alpha] phosphorylation. Transfected PKA-activated p85[alpha] binds p[21.sup.ras], reducing ERK1/2 activation and VSMC proliferation in vitro. In contrast, EC proliferation inhibition by cAMP is independent from PKA modification of p85[alpha] and ERK1/2 inhibition; indeed, PKA-activated p85[alpha] did not inhibit per se ERK1/2 activation and proliferation in ECs in vitro. Interestingly, cAMP reduced both VSMC and EC apoptotic death through p85[alpha] phosphorylation. Accordingly, PKA-activated p85[alpha] triggered Akt activation, reducing both VSMC and EC apoptosis in vitro. Finally, compared with controls, vascular gene transfer of PKA-activated p85[alpha] significantly reduced neointimal formation after balloon injury in rats, without inhibiting endothelial regeneration of the injured arterial segment. In conclusions, PKA-activated p85[alpha] integrates cAMP/PKA signaling differently in VSMCs and ECs. By reducing neointimal hyperplasia without inhibiting endothelial regeneration, it exerts a protective effect against restenosis after balloon injury. smooth muscle cells; endothelial cells; adenosine 3',5'-cyclic monophosphate; gene therapy doi: 10.1152/ajpheart.00738.2009

Chen B, Calvert AE, Cui H, Nelin LD. Hypoxia promotes human pulmonary artery smooth muscle cell proliferation through induction of arginase. Am J Physiol Lung Cell Mol Physiol 297 : L1151-L1159, 2009. First published October 2, 2009; doi: 10.1152/ajplung.00183.2009.--Vascular remodeling and smooth muscle cell proliferation are hallmark pathogenic features of pulmonary artery hypertension (PAH). Alterations in the metabolism of L-arginine via arginase and nitric oxide synthase play a critical role in the endothelial dysfunction seen in PAH. L-arginine metabolism by arginase produces L-ornithine and urea. L-ornithine is a precursor for polyamine and proline synthesis, ultimately leading to an increase in cellular proliferation. Given the integral role of the smooth muscle layer in the pathogenesis of hypoxia-induced PAH, we hypothesized that hypoxia would increase cellular proliferation via arginase induction in human pulmonary artery smooth muscle cells (hPASMC). We found that arginase II mRNA and protein expression were significantly increased in cultured hPASMC exposed to 1% [O.sub.2] for 24 and 48 h, which coincided with an increase in arginase activity at 48 h. There were no hypoxia-induced changes in levels of arginase I mRNA or protein in cultured hPASMC. Exposure to hypoxia resulted in more than one and a half times as many viable ceils after 120 h than normoxic exposure. The addition of the arginase inhibitor, S-(2-boronoethyl)-L-cysteine, completely prevented both the hypoxia-induced increase in arginase activity and proliferation in hPASMC. Furthermore, transfection of small interfering RNA (siRNA) targeting arginase II in hPASMC resulted in knockdown of arginase II protein levels and complete prevention of the hypoxia-induced cellular proliferation. These data support our hypothesis that hypoxia increases proliferation of hPASMC through the induction of arginase II. pulmonary hypertension; L-arginine; vascular remodeling doi: 10.1152/ajplung.00183.2009.

Li R, Maminishkis A, Banzon T, Wan Q, Jalickee S, Chen S, Miller SS. IFN[gamma] regulates retinal pigment epithelial fluid transport. Am J Physiol Cell Physiol 297: C1452-C1465, 2009. First published September 30, 2009; doi:l0.1152/ajpcell.00255.2009.--The present experiments show that IFN[gamma] receptors are mainly localized to the basolateral membrane of human retinal pigment epithelium (RPE). Activation of these receptors in primary cultures of human fetal RPE inhibited cell proliferation and migration, decreased RPE mitochondrial membrane potential, altered transepithelial potential and resistance, and significantly increased transepithelial fluid absorption. These effects are mediated through JAK-STAT and p38 MAPK signaling pathways. Second messenger signaling through cAMP-PKA pathway- and interferon regulatory factor-1-dependent production of nitric oxide/cGMP stimulated the CFTR at the basolateral membrane and increased transepithelial fluid absorption. In vivo experiments using a rat model of retinal reattachment showed that IFN[gamma] applied to the anterior surface of the eye can remove extra fluid deposited in the extracellular or subretinal space between the retinal photoreceptors and RPE. Removal of this extra fluid was blocked by a combination of PKA and JAK-STAT pathway inhibitors injected into the subretinal space. These results demonstrate a protective role for IFN[gamma] in regulating retinal hydration across the outer blood-retinal barrier in inflammatory disease processes and provide the basis for possible therapeutic interventions. interferon; inflammation; CFTR ; JAK-STAT; cAMP; cGMP; proliferation doi: 10.1152/ajpcell.00255.2009

Bellou S, Hink MA, Bagli E, Panopoulou E, Bastiaens PI, Murphy C, Fotsis T. VEGF autoregulates its proliferative and migratory ERK1/2 and p38 cascades by enhancing the expression of DUSP1 and DUSP5 phosphatases in endothelial cells. Am J Physiol Cell Physiol 297: C1477-C1489, 2009. First published September 9, 2009; doi: 10.1152/ajpcell.00058.2009.--Vascular endothelial growth factor (VEGF) is a key angiogenic factor that regulates proliferation and migration of endothelial cells via phosphorylation of extracellular signal-regulated kinase-1/2 (ERK1/2) and p38, respectively. Here, we demonstrate that VEGF strongly induces the transcription of two dual-specificity phosphatase (DUSP) genes DUSP1 and DUSP5 in endothelial cells. Using fluorescence microscopy, fluorescence lifetime imaging (FLIM), and fluorescence cross-correlation spectroscopy (FCCS), we found that DUSP1/mitogen-activated protein kinases phosphatase-1 (MKP-1) was localized in both the nucleus and cytoplasm of endothelial cells, where it existed in complex with p38 (effective dissociation constant, [K.sup.eff.sub.D], values of 294 and 197 nM, respectively), whereas DUSP5 was localized in the nucleus of endothelial cells in complex with ERK1/2 ([K.sup.eff.sub.D] 345 nM). VEGF administration affected differentially the [K.sup.eff.sub.D] values of the DUSP1/p38 and DUSP5/ERK1/2 complexes. Gain-of-function and lack-of-function approaches revealed that DUSP1/MKP-1 dephosphorylates primarily VEGF-phosphorylated p38, thereby inhibiting endothelial cell migration, whereas DUSP5 dephosphorylates VEGF-phosphorylated ERK1/2 inhibiting proliferation of endothelial cells. Moreover, DUSP5 exhibited considerable nuclear anchoring activity on ERK1/2 in the nucleus, thereby diminishing ERK1/2 export to the cytoplasm decreasing its further availability for activation. vascular endothelial growth factors; extracellular signal regulated kinase-1/2; p38; dual-specificity phosphatase 1/mitogen-activated protein kinase phosphatase-1; dual-specificity phosphatase-5 doi: 10.1152/ajpcell.00058.2009

Henney NC, Li B, Elford C, Reviriego P, Campbell AK, Wann KT, Evans BA. A large-conductance (BK) potassium channel sub-type affects both growth and mineralization of human osteoblasts. Am J Physiol Cell Physiol 297: Cc1397-C1408, 2009. First published September 23, 2009; doi: 10.1152/ajpcell.00311.2009.--The pharmacology of the large-conductance [K.sup.+] (BK) channel in human osteoblasts is not well defined, and its role in bone is speculative. Here we assess BK channel properties in MG63 cells and primary human osteoblasts and determine whether pharmacological modulation affects cell function. We used RT-PCR and patch-clamp methods to determine the expression of BK channel subunits and cell number assays in the absence and presence of BK channel modulators. RT-PCR showed the presence of KCNMA1, KCNMB1, KCNMB2, KCNMB3, and KCNMB4 subunits. The BK channel was voltage dependent, with a mean unitary conductance of 228.8 pS (n = 10) in cell-attached patches (140 mM [K.sup.+]/140 mM [K.sup.+]) and a conductance of 142.5 pS (n = 16) in excised outside-out and 155 pS (n = 6) in inside-out patches in 3 mM [K.sup.+]/140 mM [K.sup.+]. The selectivity ratio (ratio of [K.sup.+] to [Na.sup.+] permeability) was 15:1. The channel was blocked by tetraethylammonium (TEA, 0.3 mM), iberiotoxin (5-60 nM), tetrandrine (5-30 [micro]M), and paxilline (10 [micro]M) and activated by isopimaric acid (20 [micro]M). BK channel modulators affected MG63 cell numbers: TEA and tetrandrine significantly increased cell numbers at low concentrations (3 mM and 3 [micro]M, respectively) and reduced cell numbers at higher concentrations (>10 mM and >10 [micro]M, respectively). Neither iheriotoxin (20-300 nM) nor slotoxin (300 nM) affected cell numbers. The increase in cell numbers by TEA was blocked by isopimafic acid. TEA (0.1-3.0 mM) significantly increased mineralization in primary osteoblasts. In conclusion, the BK channel has a distinctive pharmacology and is thus a target for therapeutic strategies aimed at modulating osteoblast proliferation and function. MG63 cells; bone; potassium current; tetrandrine doi: 10.1152/ajpcell.00311.2009

The involvement of caveolin-1 in the regulation of embryonic stem (ES) cell growth by epidermal growth factor (EGF) is by no means clear cut. Thus we examined the relationship between EGF and caveolin-1 in mouse ES cell migration and proliferation. The results revealed that EGF increased Src, caveolin-1, focal adhesion kinase (FAK), Akt, and extra-cellular signal-regulated kinase-1/2 (ERK) phosphorylation levels. Especially, phosphorylation of caveolin-1 is attenuated by AG1478, herbimycin A (tyrosine kinase inhibitors), and pyrazolopyrimidine 2 (PP2, Src inhibitor) and EGF-induced ERK activation was blocked by PP2, methyl-[beta]-cyclodextrin (M[beta]CD), caveolin-1 small interfering RNA (siRNA), LY-294002 [phosphoinositol-3 kinase inhibitor (PI3K)], and Akt inhibitor. In addition, EGF promoted the cell migration, which was attenuated by PP2, caveolin-I siRNA, FAK siRNA, LY-294002, Akt inhibitor, and PD-98059. EGF also increased matrix metalloproteinase (MMP-2) expression levels and EGF-induced MMP2 expression was inhibited by caveolin-1 siRNA, FAK siRNA, LY-294002, Akt inhibitor, and PD-98059. Furthermore, EGF-induced increase of cell cycle proteins expression level and [[sup.3]H]thymidine incorporation was blocked by MMP inhibitor. EGF also significantly increases [[sup.3]H]thymidine incorporation and cell number, which were significantly blocked by AG 1478, PP2, M[beta]CD, caveolin-1 siRNA, FAK siRNA, LY-294002, and PD-98059 (ERK inhibitor). EGF-induced increase of protooncogenes (c-fos, c-myc, and c-Jun) and cell cycle regulatory proteins (cyclin D1, CDK4, cyclin E, and CDK2) expression levels were also attenuated by caveolin-1 siRNA and FAK siRNA. In conclusion, these results demonstrated that EGF-induced DNA synthesis and cell migration are mediated by caveolin-1, which is activated by Src, FAK, PI3K/Akt, ERK, and MMP-2 signals in mouse ES cells. mouse embryonic stem cells; epidermal growth factor; caveolin-1; phosphoinositol-3 kinase/Akt; mitogen-activated protein kinases; cell proliferation doi: 10.1152/ajpcell.00121.2009

The opioid growth factor (OGF) regulates cell proliferation of human cancer cells through the cyclin-dependent kinase inhibitory pathway, with mediation of this action by the OGF receptor (OGFr). The ubiquity of the OGF-OGFr axis in human cancer is unknown. We used 31 human cancer cell lines, representative of more than 90% of neoplasias occurring in humans, and found that OGF and OGFr were detected in the cytoplasm and nucleus by immunohistochemistry. The addition of OGF to cultures depressed cell number up to 41%, whereas naltrexone (NTX) increased cell proliferation by up to 44%, a total of 85% in the modulating capacity for the OGF-OGFr axis. Neutralization of OGF by specific antibodies led to a marked increase in cell number. Knockdown of OGFr by OGFr-siRNA resulted in a significant increase in the number of cells, even in the face of the addition of exogenous OGF. The cultures to which NTX was added and subjected to OGFr-siRNA were similar to those with OGF-siRNA alone. The OGF-OGFr axis, a physiological determinant of cell-proliferative activity, is a ubiquitous feature of human cancer cells. The identification of this native biological system in neoplasia may be important in understanding the pathophysiology of neoplasia, and in designing treatment modalities that utilize the body's own chemistry. siRNA; immunohistochemistry; tissue culture; naltrexone; opioid antagonists doi: 10.1152/ajpregu.00414.2009

20-Hydroxyeicosatetraenoic acid (20-HETE) has been reported to promote mitogenicity in a variety of cell types, including renal epithelial cells. However, the signal transduction pathways activated by 20-HETE have not been fully defined. The present study evaluated the effects of 20-HETE and its more stable agonist analogs 20-hydroxyeicosa-5(Z),14(Z)-dienoic acid (5,14-20-HEDE) and N-[20-hydroxyeicosa-5(Z),14(Z)-dienoyl]glycine (5,14-20-HEDGE) on the Raf/MEK/ERK and phosphatidylinositol 3-kinase (PI3K)-Akt pathway in LLC-PK~ renal epithelial cells. 20-HETE (20 [micro]M) increased phosphorylation of Raf-1 (2.5 [+ or -] 0.2-fold), MEK 1/2 (6.3 [+ or -] 1.6-fold), and ERKI/2 (5.8 [+ or -] 0.3-fold) compared with vehicle-treated cells. Similarly, the 20-HETE analogs also strongly activated ERK1/2 in a Raf-1- and MEK 1/2-dependent manner. Moreover, 5,14-20-HEDE increased Akt phosphorylation by 2.2 [+ or -] 0.3-fold. 20-HETE and 5,14-20-HEDE also promoted activation (Y1086) of epidermal growth factor receptor (EGFR; Y1086) by 1.9 [+ or -] 0.2- and 2.5 [+ or -] 0.2-fold, respectively. These effects were completely blocked by the EGFR inhibitor EKB-569 (0.1 [micro]M). Moreover, EKB-569 (0.1 [micro]M), as well as a c-Src inhibitor, SKI-606 (0.05 [micro]M), completely abolished the 20-HETE-mediated activation of the Raf/MEK/ERK and PI3K-Akt pathways. Blockade of PKC with bisindolylmaleimide I had no effect on 20-HETE-induced ERK1/2 activation. This study demonstrated that 20-HETE activated the Raf/ MEK/ERK and Akt pathways in renal epithelial cells secondary to the activation of c-Src and EGFR. epithelial cell proliferation; epidermal growth factor receptor; cell signaling

Naltrexone (NTX) is a potent opioid antagonist that promotes cell proliferation by upregulating DNA synthesis through displacement of the tonically active inhibitory peptide, opioid growth factor (OGF) from its receptor (OGFr). To investigate how NTX enters cells, NTX was fluorescently labeled [1-(N)-fluoresceinyl NTX thiosemicarbazone; FNTX] to study its uptake by living cultured cells. When human head and neck squamous cell carcinoma cell line (SCC-1) was incubated with FNTX for as little as 1 min, cells displayed nuclear and cytoplasmic staining of FNTX as determined by fluorescent deconvolution microscopy, with enrichment of fluorescent signal in the nucleus and nucleolus. The same temporal-spatial distribution of FNTX was detected in a human pancreatic cancer cell line (MIA PaCa-2), African green monkey kidney cell line (COS-7), and human mesenchymal stem cells (hMSCs). FNTX remained in cells for as long as 48 h. FNTX was internalized in SCC-1 cells when incubation occurred at 4[degrees]C, with the signal being comparable to that recorded at 37[degrees]C. A 100-fold excess of NTX or a variety of other opioid ligands did not alter the temporal-spatial distribution of FNTX. Neither fluorescein-labeled dextran nor fluorescein alone entered the cells. To study the effect of FNTX on DNA synthesis, cells incubated with FNTX at concentrations ranging from [10.sup.-5] to [10.sup.-8] M had a 5-bromo-2'-deoxyuridine index that was 39-82% greater than for vehicletreated cells and was comparable to that of unlabeled NTX (37-70%). Taken together, these results suggested that NTX enters cells by passive diffusion in a nonsaturable manner. naltrexone; transport; diffusion; tissue culture: cell proliferation

The development of pancreatic fibrosis has been shown to be a major component in several diseases of the pancreas including pancreatic cancer, chronic pancreatitis, and type 2 diabetes mellitus, but its actual role in the progression of these disorders is still unknown. This fibrosis is characterized by stromal expansion and the excessive deposition of extracellular matrix (ECM) that replaces pancreatic tissue. This eventually leads to dysregulation of ECM turnover, production of cytokines, restriction of blood flow, and often exocrine and endocrine insufficiencies. Activated pancreatic stellate cells (PSCs) have been identified as key mediators in the progression of pancreatic fibrosis, serving as the predominant source of excess ECM proteins. Previously, we found that overexpression of the growth factor heparin-binding epidermal growth factor-like growth factor (HB-EGF) in pancreatic islets led to intraislet fibrosis. HB-EGF binds to and activates two receptors, epidermal growth factor receptor (EGFR) and ErbB4, as well as heparin moieties and CD9/DRAP27. To understand the mechanism underlying the induction of fibrogenesis by HB-EGF, we utilized a hypomorphic allele of Egfr, the Waved-2 allele, to demonstrate that EGFR signaling regulates fibrogenesis in vivo. Using an in vitro cell migration assay, we show that HB-EGF regulates both chemoattraction and stimulation of proliferation of PSCs via EGFR activation, heparin-binding epidermal growth factor-like growth factor; pancreatic stellate cells; Waved-2

Because transient receptor potential (TRP) channels have been implicated in tumor progression, we have investigated the potential role of TRPM7 channel in breast cancer cell proliferation. Under whole cell patch clamp, a [Mg.sup.2+]-inhibited cationic (MIC) current was observed in MCF-7 cells. This current was characterized by an inward current and a strong outward rectifying current that were both inhibited in a concentration-dependent manner by the presence of intracellular [Mg.sup.2+] or [Mg.sup.2+]-ATP. The inward current was reduced by [La.sup.3+], and the outward current was sensitive to 2-aminoethoxydiphenyl borate (2-APB), spermine, [La.sup.3+], and flufenamic acid. Importantly, a similar MIC current was also recorded in the primary culture of human breast cancerous epithelial cells (hBCE). Moreover, TRPM7 transcripts were found in both hBCE and MCF-7 cells. In MCF-7 cells, the MIC current was inhibited by TRPM7 small interfering RNA. Interestingly, we found that cell proliferation and intracellular [Ca.sup.2+] concentration were also reduced by TRPM7 silencing in MCF-7 cells. TRPM7 channels were also found in both human breast cancer and healthy tissues. Importantly, TRPM7 channel was overexpressed in grade III breast cancer samples associated with important Ki67 or tumor size. Our findings strongly suggest that TRPM7 is involved in the proliferative potentiality of breast cancer cells, probably by regulating [Ca.sup.2+] influx. transient receptor potential

The objective of this study was to better understand the role of transforming growth factor-[beta] (TGF-[beta]) and its primary signaling protein Smad3 in the development of intimal hyperplasia. Male Sprague-Dawley rats underwent left carotid balloon injury followed by intra-arterial infection with adenovirus-expressing Smad3 (AdSmad3). In uninfected injured arteries, endogenous Smad3 was upregulated with the expression peaking at 14 days. Moreover, in arteries infected with AdSmad3, we observed an enhancement of intimal hyperplasia and increased vascular smooth muscle cell (VSMC) proliferation. The novel finding, that TGF-[beta]/Smad3 stimulated rather than inhibited VSMC proliferation, was confirmed in cultured VSMCs infected with AdSmad3 and treated with TGF-[beta]. To identify the mechanism underlying TGF-[beta]/ Smad3-mediated VSMC proliferation, we studied the cyclin-dependent kinase inhibitor p27. Although the upregulation of Smad3 in VSMCs had no significant effect on total p27 levels, Smad3 did stimulate the phosphorylation of p27 at serine-10 as well as the nuclear export of p27, events associated with cell proliferation. Furthermore, serine-10-phosphorylated p27 was also increased in AdSmad3-infected injured rat carotid arteries, demonstrating the existence of this same mechanism in vivo. In conclusion, our findings identify a novel mechanism for the effect of TGF-[beta] on intimal hyperplasia. In the presence of elevated levels of Smad3 that develop in response to injury, TGF-[beta] stimulates smooth muscle cell proliferation through a mechanism involving the phosphorylation and nuclear export of p27. serine-10-phosphorylated p27; rat carotid balloon injury

Both estrogen ([E.sub.2]) and EGF regulate lactotrophs, and we recently demonstrated that EGF phosphorylates S118 on estrogen receptor-[alpha] (ER[alpha]) and requires ER[alpha] to stimulate prolactin (PRL) release. However, the interactions between ligand-occupied ER[alpha] and activated ErbB1 and its impact on lactotroph function are unknown. Using rat GH3 lactotrophs, we found that both [E.sub.2] and EGF independently stimulated proliferation and PRL gene expression. Furthermore, their combination resulted in an enhanced stimulatory effect on both cell proliferation and PRL gene expression. Inhibitors of ER as well as ErbB1 blocked the combined effects of E2 and EGF. Pretreatment with UO126 abolished the combined effects, demonstrating Erk1/2 requirement. Although bidirectionality in ER-ErbB1 cross-talk is a well-accepted paradigm, interestingly in lactotrophs, ErbB1 kinase inhibitor failed to block the effect of E2 on proliferation and stimulation of PRL gene expression, suggesting that ER does not require ErbB1 to mediate its effects. Furthermore, E2 did not affect the ability of EGF to induce c-Fos expression or modulate AP-1 activity. However, both E2 and EGF combine to enhance S118 phosphorylation of ER[alpha], leading to enhanced E2-mediated estrogen response element transactivation. Taken together, our results suggest that, in lactotrophs, activated ErbB1 phosphorylates ER[alpha] to enhance the stimulatory effect of E2, thereby providing the molecular basis by which EGF amplifies the response of [E.sub.2]. lactotrophs; cell proliferation

Patients with chronic renal failure are at greater risk of developing atherosclerosis than healthy individuals, and recent data suggest that the putative uremic toxin indoxyl sulfate (IS) promotes the pathogenesis of atherosclerosis. The present study examined the effects of IS on vascular smooth muscle cells (VSMCs) with respect to reactive oxygen species (ROS), platelet-derived growth factor (PDGF) receptors, and mitogen-activated protein kinases (MAPKs). IS induced the migration and proliferation of VSMCs and synergistically enhanced their PDGF-induced migration as well as proliferation. The effects of PDGF were promoted after a 24-h incubation with IS despite the absence of IS during PDGF stimulation. Intracellular ROS levels were increased in the presence of IS, and PDGF-dependent ROS production was augmented by a prior 24-h incubation with IS even in the absence of IS during PDGF stimulation. These data suggest that IS increases the sensitivity of VSMCs to PDGF. IS also phosphorylated PDGF-[beta]-receptors and upregulated PDGF-[beta] receptor but not a-receptor protein expression in the absence of exogenous PDGF. The NADPH oxidase inhibitor diphenylene iodonium blocked IS-dependent increase in receptor expression. Administration of IS to nephrectomized rats also elevated receptor protein expression in arterial VSMCs. Inhibitors of NADPH oxidase, PDGF-[beta] receptors, extracellular-regulated protein kinase (ERK), and p38 MAPK all inhibited IS-induced VSMCs migration and proliferation. Taken together, these findings indicate that IS induces the migration as well as proliferation of VSMCs through PDGF-[beta] receptors and that ROS generation is critically involved in this process, which promotes the development of atherosclerosis. uremic toxin; chronic renal failure; atherosclerosis; reactive oxygen species; platelet-derive growth factor-[beta]

We previously showed that an increase in the peroxisome proliferator-activated receptor-[delta] (PPAR[delta]), together with subsequent induction of inducible nitric oxide synthase (iNOS) by beraprost (BPS), inhibits aortic smooth muscle cell proliferation. Herein, we delineated the mechanisms of the antiproliferative effects of BPS through the induction of p21/p27. BPS concentration dependently induced the p21/p27 promoter- and consensus cAMP-responsive element (CRE)-driven luciferase activities, which were significantly suppressed by blocking PPAR[delta] activation. Surprisingly, other than altering the CRE-binding protein (CREB), BPS-mediated PPAR[delta] activation increased nuclear localization of the CREB-binding protein (CBP), a coactivator, which was further confirmed by chromatin immunoprecipitation. Furthermore, novel functional PPAR-responsive elements (PPREs) next to CREs in the rat p21/p27 promoter regions were identified, where PPAR[delta] interacted with CREB through CBP recruitment. BPS-mediated suppression of restenosis in mice with angioplasty was associated with p21/p27 induction. Herein, we demonstrate for the first time that BPS-mediated PPAR[delta] activation enhances transcriptional activation of p21/p27 by increasing CBP nuclear translocation, which contributes to the vasoprotective action of BPS. cAMP-responsive element; cAMP-responsive element-binding protein; cAMP-responsive element-binding protein-binding protein; antiproliferation

Stem cells, their niches, and their relationship to cancer are under intense investigation. Because tumors and metastases acquire self-renewing capacity, mechanisms for their establishment may involve cell-cell interactions similar to those between stem cells and stem cell niches. On the basis of our studies in Caenorhabditis elegans, we introduce the concept of a 'latent niche' as a differentiated cell type that does not normally contact stem cells nor act as a niche but that can, under certain conditions, promote the ectopic self-renewal, proliferation, or survival of competent cells that it inappropriately contacts. Here, we show that ectopic germ-line stem cell proliferation in C. elegans is driven by a latent niche mechanism and that the molecular basis for this mechanism is inappropriate Notch activation. Furthermore, we show that continuous Notch signaling is required to maintain ectopic germ-line proliferation. We highlight the latent niche concept by distinguishing it from a normal stem cell niche, a premetastatic niche and an ectopic niche. One of the important distinguishing features of this mechanism for tumor initiation is that it could operate in the absence of genetic changes to the tumor cell or the tumor-promoting cell. We propose that a latent niche mechanism may underlie tumorigenesis and metastasis in humans. Caenorhabditis elegans | Delta/Serrate/LAG-2 | Notch | stem cell | Pro phenotype

A long-standing question in biology is whether there is an intrinsic mechanism for coordinating growth and the cell cycle in metazoan cells. We examined cell size distributions in populations of lymphoblasts and applied a mathematical analysis to calculate how growth rates vary with both cell size and the cell cycle. Our results show that growth rate is size-dependent throughout the cell cycle. After initial growth suppression, there is a rapid increase in growth rate during the [G.sub.1] phase, followed by a period of constant exponential growth. The probability of cell division varies independently with cell size and cell age. We conclude that proliferating mammalian cells have an intrinsic mechanism that maintains cell size.

Follistatin (FS) inhibits several members of the TGF-[beta] superfamily, including myostatin (Mstn), a negative regulator of muscle growth. Mstn inhibition by FS represents a potential therapeutic approach of muscle atrophy. The aim of our study was to investigate the mechanisms of the FS-induced muscle hypertrophy. To test the role of satellite cells in the FS effect, we used irradiation to destroy their proliferative capacity. FS overexpression increased the muscle weight by about 37% in control animals, but the increase reached only 20% in irradiated muscle, supporting the role of cell proliferation in the FS-induced hypertrophy. Surprisingly, the muscle hypertrophy caused by FS reached the same magnitude in Mstn-KO as in WT mice, suggesting that Mstn might not be the only ligand of FS involved in the regulation of muscle mass. To assess the role of activin (Act), another FS ligand, in the FS-induced hypertrophy, we electroporated FSI-I, a FS mutant that does not bind Act with high affinity. Whereas FS electroporation increased muscle weight by 32%, the muscle weight gain induced by FSI-I reached only 14%. Furthermore, in Mstn-KO mice, FSI-I overexpression failed to induce hypertrophy, in contrast to FS. Therefore, these results suggest that Act inhibition may contribute to FS-induced hypertrophy. Finally, the role of Act as a regulator of muscle mass was supported by the observation that ActA overexpression induced muscle weight loss (-15%). In conclusion, our results show that satellite cell proliferation and both Mstn and Act inhibition are involved in the FS-induced muscle hypertrophy.

BACKGROUND: Fetal exposure to environmental estrogens may contribute to hypofertility and/or to testicular germ cell cancer. However, many of these xenoestrogens have only a weak affinity for the classical estrogen [...]

The development of metastases is an extended and inefficient process involving multiple steps. The last of these involves the growth of micrometastases into macroscopic tumors. We show here that intravenously injected, nonmetastatic cancer cells cease proliferating after extravasating into the parenchyma of the lungs; this response is attributable to the cells inability to trigger adhesion-related signaling events when they are scattered sparsely within the extracellular matrix (ECM) of the parenchyma. We recapitulate this situation by culturing these nonmetastatic cells at low seeding density in ECM-derived gels in vitro, in which they undergo cell-cycle arrest resulting, in part, from insufficient activation of focal adhesion kinase (FAK). Metastatic cancer cells, in contrast, show sufficient FAK activation to enable their proliferation within ECM gels in vitro and continue cell-cycle progression within the lung parenchyma in vivo. Activation of FAK in these metastatic cells depends on expression of the [[beta].sub.1] subunit of integrins, and proliferation of these cells after extravasation in the lungs is diminished by knocking down the expression of either FAK or integrin [[beta].sub.1]. These results demonstrate the critical role of integrin [[beta].sub.1]-FAK signaling axis in controlling the initial proliferation of micrometastatic cancer cells disseminated in the lungs. dormancy | micrometastasis | cell-matrix adhesion

Optimizing the function and proliferative capacity of stem cells is essential to maximize their therapeutic benefits. High glucose concentrations are known to have detrimental effects on many cell types. We hypothesized that human mesenchymal stem cells (hMSCs) cultured in high glucose-containing media would exhibit diminished proliferation and attenuated production of VEGF, hepatocyte growth factor (HGF), and FGF2 in response to treatment with TNF-[alpha], LPS, or hypoxia, hMSCs were plated in medium containing low (5.5 mM) and high (20 mM or 30 mM) glucose concentrations and treated with TNF-[alpha], LPS, or hypoxia. Supernatants were collected at 24 and 48 h and assayed via ELISA for VEGF, HGF, and FGF2. In addition, hMSCs were cultured on 96-well plates at the above glucose concentrations, and proliferation at 48 h was determined via bromo-2'-deoxy-uridine (BrdU) incorporation. At 24 and 48 h, TNF-[alpha], LPS, and hypoxia-treated hMSCs produced significantly higher VEGF, HGF, and FGF2 compared with control. Hypoxia-induced VEGF production by hMSCs was the most pronounced change over baseline. At both 24 and 48 h, glucose concentration did not affect production of VEGF, HGF, or FGF2 by untreated hMSCs and those treated with TNF-[alpha] LPS, or hypoxia. Proliferation of hMSCs as determined via BrdU incorporation was unaffected by glucose concentration of the media. Contrary to what has been observed with other cells, hMSCs may be resistant to the short-term effects of high glucose. Ongoing efforts to characterize and optimize ex vivo and in vivo conditions are critical if the therapeutic benefits of MSCs are to be maximized. transplantation; preconditioning; inflammation; paracrine effects

prohibitin ring complexes in the mitochondrial inner membrane regulate cell proliferation as well as the dynamics and function of mitochondria. Although prohibitins are essential in higher eukaryotes, prohibitin-deficient yeast cells are viable and exhibit a reduced replicative life span. Here, we define the genetic interactome of prohibitins in yeast using synthetic genetic arrays, and identify 35 genetic interactors of prohibitins (GEP genes) required for cell survival in the absence of prohibitins. Proteins encoded by these genes include members of a conserved protein family, Ups1 and Gep1, which affect the processing of the dynamin-like GTPase Mgm1 and thereby modulate cristae morphogenesis. We show that Ups1 and Gep1 regulate the levels of cardiolipin and phosphatidylethanolamine in mitochondria in a lipid-specific but coordinated manner. Lipid profiling by mass spectrometry of GEP-deficient mitochondria reveals a critical role of cardiolipin and phosphatidylethanolamine for survival of prohibitin-deficient cells. We propose that prohibitins control inner membrane organization and integrity by acting as protein and lipid scaffolds.

The Mst1 and Mst2 protein kinases are the mammalian homologs of hippo, a major inhibitor of cell proliferation in Drosophila. Mst1 is most abundant in lymphoid tissues. Mice lacking Mst1 exhibit markedly reduced levels of the Mst1 regulatory protein Nore1B/ RAPL in lymphoid cells, whereas Mst2 abundance is unaltered. Mst1-null mice exhibit normal T cell development but low numbers of mature naive T cells with relatively normal numbers of effector/ memory T cells. In vitro, the Mst1-deficient naive T cells exhibit markedly greater proliferation in response to stimulation of the T cell receptor whereas the proliferative responses of the Mst1-null effector/memory T cell cohort is similar to wild type. Thus, elimination of Mst1 removes a barrier to the activation and proliferative response of naive T cells. The levels of Mst1 and Nore1B/RAPL in wild-type effector/memory T cells are approximately 10% those seen in wild-type naive T cells, which may contribute to the enhanced proliferative responses of the former. Freshly isolated Mst1-null T cells exhibit high rates of ongoing apoptosis, a likely basis for their low numbers in vivo; they also exhibit defective clustering of LFA-1, as previously observed for Nore1B/RAPL-deficient T cells. Among known Mst1 substrates, only the phosphorylation of the cell cycle inhibitory proteins MOBKL1A/B is lost entirely in TCR-stimulated, Mst1-deficient T cells. Mst1/2-catalyzed MOBKL1A/B phosphorylation slows proliferation and is therefore a likely contributor to the anti-proliferative action of Mst1 in naive T cells. The Nore1B/RAPL-Mst1 complex is a negative regulator of naive T cell proliferation. Mob1 | Mst1 knockout | RAPL | hippo | Stk4

Vitamin D has pleiotropic effects that go beyond its traditional role in calcium homeostasis. Hundreds of genes with vitamin D receptor response elements directly or indirectly influence cell cycling and proliferation, differentiation, and apoptosis. Vitamin D compounds also have effects on cell function that are nongenomic. The noncalcemic actions of vitamin D influence normal and pathological cell growth, carcinogenesis, immune function, and cardiovascular physiology. This review examines many of the various mechanisms by which vitamin D alters cellular growth and differentiation and explores cell-specific factors that influence responsiveness to vitamin D.

Titin (also called connectin) is a major protein in sarcomere assembly as well as providing elastic return of the sarcomere postcontraction in cardiac and striated skeletal muscle tissues. In addition, it has been speculated that titin is associated with nuclear functions, including chromosome and spindle formation, and regulation of muscle gene expression. In the present study, a short isoform of titin was detected in a human osteoblastic cell line, MG-63 cells, by both immunostaining and Western blot analysis. Confocal images of titin staining showed both cytoplasmic and nuclear localization in a punctate pattern. Therefore, we hypothesized that human titin may contain a nuclear localization signal (NLS). A functional NLS, 200-PAKKTKT-206, located in a low-complexity, titin-specific region between Z2 and Z repeats, was found by sequentially deleting segments of the N[H.sub.2]-terminal sequence in conjunction with an enhanced green fluorescent protein reporter system and confirmed by site-directed mutagenesis. Overexpression of titin's amino terminal fragment (Z1Z2Zr) in human osteoblasts (MG-63) increased cell proliferation by activating the Wnt/[beta]-catenin pathway. RT-PCR screens of tissue panels demonstrated that residues 1-206 were ubiquitously expressed at low levels in all tissues and cell types analyzed. Our data implicate a dual role for titin's amino terminal region, i.e., a novel nuclear function promoting cell division in addition to its known structural role in Z-line assembly. Wnt; catenin; osteoblast