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Grainyhead-like 2 (Grhl2) is a transcription factor regulating cell adhesion genes. Grhl2 acts as an epithelial–mesenchymal transition suppressor, and it is a proto-oncogene involved in estrogen-stimulated breast cancer proliferation. However, its expression during ovarian hormone–dependent mammary ductal development remains obscure. We here examined Grhl2 expression in the mammary gland of normal and steroid-replaced ovariectomized mice. Grhl2 protein signals were detected in both the mammary luminal epithelial and myoepithelial nuclei. The ratio and density of Grhl2-positive nuclei increased after the onset of puberty and progressed with age, whereas Grhl2-negative epithelial cells were detected in mature ducts. Claudin 3, claudin 4, claudin 7, and E-cadherin gene expression in the mammary gland was upregulated, and their expression was highly correlated with Grhl2 gene expression. Furthermore, Grhl2 mRNA expression and ductal lumen width were significantly increased by the combined treatment of estrogen and progesterone compared with estrogen alone. These results suggest that Grhl2 expressed in the luminal epithelial and myoepithelial cells from the early phase of ductal development, controlling the expression of cell adhesion molecules to establish functional ducts

Oxytocin, a posterior pituitary hormone, causes the contraction of the mammary myoepithelial cells that surround the acini. This ejects milk from the acini into the primary mammary ducts. The milk ejection responses by oxytocin have not yet been exactly evaluated in mice. Thus, we present a novel method for quantitatively evaluating oxytocin-induced milk ejection in anesthetized lactating mice. We cannulated the mammary duct, administered oxytocin intraperitoneally or intravenously, and collected and measured the ejected milk. Intraperitoneal oxytocin administration (150 mU) induced continuous but oscillatory milk ejection. Repeated intravenous administration of 1.5 mU of oxytocin elicited repeated transient milk ejection. The volume of the ejected milk as a proportion of the stored volume just before each ejection (rather than ejection volume itself) was an expedient and reliable parameter representing the potency of ejection. The oxytocin sensitivity of mice at day 18 of lactation was determined from a sigmoidal dose–response curve as ED50 ≈ 2.69 mU. Based on this dose–response relationship, the specific activity of the oxytocin receptor agonists (Thr4, Gly7)-oxytocin and WAY 267464 were estimated as 976 and 6.87 U/mg, respectively. The assay presented here could be useful for physiological and pharmacological investigations of oxytocin-induced milk ejection.

Membrane transporters are essential for the synthesis and secretion of milk. We are at a transition point; in the twentieth century, most transport studies were carried out by following the transported molecule into the cell and examining all the ways that transport could be perturbed by the environment, allowing functional descriptions of most transport processes. We now have powerful molecular tools so that the molecular nature of the transporters as well as the mRNAs from which they are transcribed can be studied; in addition, antibodies to the proteins allow the transporters to be localized within the cell so that real cellular pathways can be discerned. So that our current knowledge can be of most use to the scientific community we use mRNA expression levels from two datasets representing complete catalogues of expressed genes in the lactating mammary gland of the human and the mouse. These expression levels are compared with expression levels in the colostral and transitional milk phases in the case of human milk and with midpregnancy expression levels in the mouse. The data provide a molecular framework, which should greatly facilitate future elucidation of transporters and their function in the cellular compartments involved in the secretion of milk.

It is well documented that estrogen is predominant inducer of hepatocyte growth factor (HGF) in a variety of cell types. However, the effect of progesterone (P) remains to be elusive. Thus, in the present study, we examined the effect of P and combined effect of P and 17β-estradiol (E2) on HGF expression and production in 3T3-L1 fibroblastic preadipocytes and mature adipocytes, as a model of stromal cells. Northern blot analysis showed that hgf mRNA expressed in preadipocytes was notably higher than that of mature adipocytes, and increased by treatment of preadipocytes with E2 or 10 nM P, but not with 1,000 nM P. The E2-induced hgf mRNA expression was enhanced by 10 nM P, but suppressed by 1,000 nM P. Western blot analysis revealed that biological active forms of HGF protein was found in the preadipocyte culture medium, while the lesser amount of HGF precursor protein was detected in the mature adipocyte culture medium. The amounts of HGF were changed dependently on the hgf mRNA expression levels. These results indicate that HGF production is intricately regulated by E2 and P at the transcriptional levels in 3T3-L1 cells, and may explain the changes in the HGF production during the mammary gland development, especially decrease in HGF expression during pregnancy when P concentration is high.

The Cl− secretion via Ca2+-activated Cl− channel (CaCC) is critical for fluid secretion in exocrine glands like the salivary gland. Also in the mammary gland, it has been hypothesized that CaCC plays an important role in the secretion of Cl− and aqueous phase of milk. However, there has been no evidence for the functional expression of CaCC in native mammary secretory (MS) cells of lactating animals. We therefore assessed membrane current in MS cells that were freshly isolated from lactating mice using whole cell patch-clamp techniques. In MS cells, we detected CaCC current that exhibited the following characteristics: 1) Ca2+-dependent activation at the concentrations of submicromolar range; 2) voltage-dependent activation; 3) slow kinetics for activation and deactivation; 4) outward rectification of the steady-state current; 5) anion permeability in the sequence of I− > NO3− > Br− > Cl− >> glutamate; 6) inhibition by Cl− channel blockers (niflumic acid, DIDS, and CaCCinh-A01). These characteristics of native CaCC current were similar to reported characteristics of heterologously expressed TMEM16A. RT-PCR analyses showed the expression of multiple CaCC channels including TMEM16A, Best1, and Best3 in the mammary glands of lactating mice. Immunohistochemical staining revealed the localization of TMEM16A protein at the apical membrane of the MS cells. Collectively, our data strongly suggest that MS cells functionally express CaCC, which is at least partly constituted by TMEM16A. The CaCC such as TMEM16A at the apical membrane of the MS cells may influence the quantity and/or quality of milk.

Anoctamin 1 (encoded by the Ano1 gene) is a Ca2+-activated Cl- channel critical to many physiological functions. It has been speculated that Ano1 expression is regulated in a tissue-dependent manner via alternative promoters. However, variation in the 5'-end sequence of mouse Ano1 (mAno1) and its tissue-dependent regulation are poorly understood. We identified a novel 5'-terminal exon (designated exon 1a) of mAno1 instead of the known 5'-terminal exon (exon 0) using 5'-rapid amplification of cDNA ends (RACE) analysis. Unexpectedly, the novel 5'-end variant mAno1Ex1a was abundantly expressed in many tissues including the salivary and mammary glands, rectum, lung, trachea and prostate. In contrast, the known variant mAno1Ex0 predominated only in male reproductive tissues such as the epididymis and testis. In a heterologous expression system, mAno1Ex0 encoded a longer protein than mAno1Ex1a, and this long isoform was abolished by a mutation in the exon 0 start codon. Moreover, the mAno1Ex0-specific N-terminal sequence was immunohistochemically detected in epididymis but not in salivary gland. Our data suggest that mAno1 expression is regulated via alternative promoters, and its transcriptional variation results in variation of the N-terminal sequence of the Ano1 protein due to the alternative translation initiation sites. These tissue-specific variations might contribute to the regulation of mAno1 expression and activity according to the physiological function of each tissue.

Hepatocyte growth factor (HGF) is a mesenchymal cell-derived factor that regulates cell growth, cell motility, and morphogenesis. Since there are conflicting reports on HGF-producing cells, we herein examined HGF activity in conditioned medium (CM) of bovine and mouse preadipocytes before and after adipogenic differentiation. CM of bovine adipocytes and mouse preadipocytes induced the morphogenesis of mammary epithelial cells that was inhibited by an NK4 HGF antagonist, whereas CM of bovine preadipocytes and mouse adipocytes did not. HGF mRNA expression was increased by a treatment with dexamethasone and isobutylmethylxanthine in bovine as well as human cells, whereas it was decreased in rodent cells. It was unfortunate that HGF gene promoter activity failed to reflect HGF mRNA expression in these cells. After actinomycin D treatment, expression of HGF mRNA remained stable in pre- and differentiated bovine adipocytes and mouse preadipocytes, whereas rapidly decreased in mouse-differentiated adipocytes. These results indicate that expression and production of HGF are regulated in a species-specific adipogenic differentiation-dependent manner and suggest that the decrease in HGF mRNA in mouse differentiated adipocytes is, at least in part, mediated by differentiation-dependent loss of its stability.

K+channels in mammary secretory (MS) cells are believed to play a role in transcellular electrolyte transport and thus determining ionic composition of the aqueous phase of milk. However, direct evidence for specific K+channel activity in native MS cells is lacking at the single-cell level. Here, we show for the first time that an inwardly rectifying K+(Kir) channel is functionally expressed in fully differentiated MS cells that were freshly isolated from the mammary gland of lactating mice. Using the standard whole cell patch-clamp technique, we found that mouse MS cells consistently displayed a K+current, whose electrophysiological properties are similar to those previously reported for Kir2.x channels, particularly Kir2.1: 1) current-voltage relationship with strong inward rectification, 2) slope conductance approximately proportional to the square root of external K+concentration, 3) voltage- and time-dependent and high-affinity block by external Ba2+, and 4) voltage-dependent inhibition by external Cs+. Accordingly, RT-PCR analysis revealed the gene expression of Kir2.1, but not Kir2.2, Kir2.3, and Kir2.4, in lactating mouse mammary gland, and immunohistochemical staining showed Kir2.1 protein expression in the secretory cells. Cell-attached patch recordings from MS cells revealed that a 31-pS K+channel with strong inward rectification was likely active at the resting membrane potential. Collectively, the present work demonstrates that a functional Kir2.1-like channel is expressed in lactating mouse MS cells. We propose that the channel might be involved, at least in part, in secretion and/or preservation of ionic components of milk stored into the lumen of these cells.

It is well known that retinoic acid (RA) suppresses adipogenesis, although there are some contradicting reports. In this study, we examined the effect of extracellular glucose on RA-induced suppression of adipogenesis in 3T3L1 cell culture. When the cells were cultured in normal glucose medium (NG), the addition of RA suppressed lipid accumulation. However, when cultured in high glucose medium (HG), addition of RA to the cells enhanced lipid accumulation. These changes were accompanied by parallel alterations in fatty acid synthase (FAS) and sterol regulatory element-binding protein (SREBP)-1 gene expression. Transfection of SREBP-1 siRNA suppressed RA-induced enhancement of lipid accumulation and FAS expression in the cells cultured with HG. Transfection of the nuclear form of SREBP-1a cDNA into the cells cultured with NG inhibited RA-induced suppression of lipid accumulation and FAS expression. Moreover, RA- and HG-induced SREBP-1a expression occurred at the early phase of adipogenesis and was dependent on glucocorticoid to induce liver X receptor (LXR) β, peroxisomal proliferator-activated receptor (PPAR) γ and retinoid X receptor (RXR), the key nuclear factors influencing the SREBP-1a gene expression. These results suggest that RA suppresses and enhances lipid accumulation through extracellular glucose concentration-dependent modulation of SREBP-1 expression.

Glycoconjugates in the olfactory system play critical roles in neuronal formation, and α1-2 fucose (α1-2Fuc) glycan mediates neurite outgrowth and synaptic plasticity. Histochemical findings of α1-2Fuc glycan in the mouse olfactory system detected using Ulex europaeus agglutinin-I (UEA-I) vary. This study histochemically assessed the main olfactory and vomeronasal pathways in male and female ICR and C57BL/6J mice aged 3-4 months using UEA-I. Ulex europaeus agglutinin-I reacted with most receptor cells arranged mainly at the basal region of the olfactory epithelium. The olfactory nerve layer and glomerular layer of the main olfactory bulb were speckled with positive UEA-I staining, and positive fibers were scattered from the glomerular to the internal plexiform layer. The lateral olfactory tract and rostral migratory stream were also positive for UEA-I. We identified superficial short-axon cells, interneurons of the external plexiform layer, external, middle and internal tufted cells, mitral cells and granule cells as the origins of the UEA-I-positive fibers in the main olfactory bulb. The anterior olfactory nucleus, anterior piriform cortex and olfactory tubercle were negative for UEA-I. Most receptor cells in the vomeronasal epithelium and most glomeruli of the accessory olfactory bulb were positive for UEA-I. Our findings indicated that α1-2Fuc glycan is located within the primary and secondary, but not the ternary, pathways of the main olfactory system, in local circuits of the main olfactory bulb and within the primary, but not secondary, pathway of the vomeronasal system.

Proinsulin C-peptide shows beneficial effects on microvascular complications of Type 1 diabetes. However, the possible occurrence of membrane C-peptide receptor(s) has not been elucidated. The aim of this study was to identify and characterize membrane proteins to which C-peptide binds. The enzyme α-enolase was co-immunoprecipitated with C-peptide after chemical cross-linking to HL-60 cell surface proteins and identified by mass spectrometry. Recombinant α-enolase activity was modulated by C-peptide, with a significant decrease in K(m) for 2-phosphoglycerate without affecting V(max). The enzyme modulation by C-peptide was abolished when C-terminal basic lysine residue (K434) of the enzyme was replaced by neutral alanine or acidic glutamate, but not with basic arginine. The enzyme modulation by C-peptide was reproduced with the C-peptide fragments containing glutamate corresponding to position 27 (E27) of the full-length C-peptide. Addition of a lysine analogue to the assay and A31 cell culture abrogated the enzyme modulation and MAP kinase activation by C-peptide, respectively. The results indicate that C-peptide has the capacity to activate α-enolase through a specific interaction between E27 of the peptide and K434 of the enzyme. Since α-enolase plays a role as a cell surface receptor for plasminogen, it may conceivably also serve as a receptor for C-peptide in vivo.

The thermogenic function of brown adipose tissue (BAT) is increased by norepinephrine (NE) released from sympathetic nerve endings, but the roles of NPY released along with NE are poorly elucidated. Here, we examined effect of NPY on basal and NE-enhanced thermogenesis in isolated brown adipocytes that express Y1 and Y5 receptor mRNA. Treatment of cells with NPY did not influence the basal and NE-enhanced rates of oxygen consumption and cAMP accumulation. Treatment with NPY also failed to induce ERK (Thr202/Tyr204) phosphorylation in the brown adipocytes. In contrast, treatment with NPY increased ERK phosphorylation in cultured stromal vascular cells from the BAT that express Y1 receptor mRNA. In the latter treatment with NPY also increased STAT3 (Ser727) phosphorylation. These results suggest that NPY mainly acts on stromal vascular cells in BAT and plays roles in the regulation of their gene transcription through ERK and STAT3 pathways, while NPY does not affect the thermogenic function of brown adipocytes.

B6.MRLc1(82—100) congenic mice carrying the telomeric region of lupus-prone MRL chromosome 1 develop autoimmune glomerulonephritis (GN). The GN susceptibility locus of B6.MRLc1(82—100) contains the interferon activated gene 200 (Ifi200) family, which consists of Ifi202, 203, 204, and 205. Recently, Ifi202 was suggested as a candidate gene for murine lupus. In this study, we assessed the association between Ifi200 family and GN in several disease models. We compared the expression of Ifi200 family members in 24 organs between the C57BL/6 and B6.MRLc1(82-100). The expressions of Ifi200 family members differed between strains, and the most dramatic differences appeared in Ifi202 expression. Briefly, in the blood, immune organs, lungs, and testes mRNA expression was higher in B6.MRLc1(82—100) mice. In the kidney and immune organs, only Ifi202 expression increased with the development of GN in B6.MRLc1(82—100), and significant differences from C57BL/6 were observed even before disease onset. Ifi202 expression in the kidneys of BXSB, NZB/WF1, and MRL/lpr was also significantly high in the early- and late-disease stages. Furthermore, laser microdissection-reverse-transcriptase-polymerase chain reaction analysis confirmed the high Ifi202 expression in all areas of B6.MRLc1(82—100) kidneys. In conclusion, in the Ifi200 family, Ifi202 expressions in the kidney and immune organs significantly increased with GN progression.

Mammary glands develop postnatally in response to the hypothalamic-pituitary-gonadal axis. Obesity-induced changes in the local environment, however, retard mammary gland development during late pregnancy and lactation. To clarify the effects of obesity on fundamental duct development, we compared the mammary glands of nulliparous nonpregnant obese mice fed a high-fat diet with those of lean mice fed a normal diet. Obese mice had enlarged mammary glands, reflecting fat pad size, whereas the ducts in obese mice showed a less dense distribution with less frequent branching. Additionally, the ducts were surrounded by thick collagen layers, and were incompletely lined with myoepithelium. Because leptin receptors were localized in the epithelium region and leptin that was highly expressed in the obese glands suppressed mammary epithelial cell proliferation in vitro, the present results suggest that obesity disrupts mammary ductal development, possibly by remodeling the mammary microenvironment and promoting the expression of such paracrine factors as leptin.

Background Proinsulin C-peptide shows ameliorative effects on diabetic complications, possibly through the production of nitric oxide (NO). On the contrary, increased local availability of NO and expression of endothelial NO synthase (eNOS) in the renal endothelium are shown to be involved in the progression of diabetic nephropathy. The aim of this study was to elucidate the effect of C-peptide and insulin as a reference on the eNOS expression in the early phase of type 1 diabetic rat kidney. Methods Type 1 diabetes in rats was produced by streptozotocin injection and some of the rats were treated with either C-peptide or insulin by the aid of an osmotic pump for 1 week. Conventional biochemical and histological analyses were performed on tissue samples. Results The diabetic rats showed hyperglycemia with over 90% reduction of endogenous insulin and C-peptide. Replacement with C-peptide or insulin resulted in recovery of weight lost, but only insulin infusion lowered plasma-glucose concentration. The eNOS protein was localized in glomeruli and endothelial cells of arterioles, and its amounts in the kidneys, but not in the lungs, of diabetic rats was increased. Replacement with C-peptide or insulin-abrogated diabetes-induced increase of renal eNOS protein. Conclusion The results indicate that C-peptide suppresses diabetes-induced abnormal renal eNOS expression, by which C-peptide may exert beneficial effects on diabetic nephropathy. Copyright © 2007 John Wiley & Sons, Ltd.

A selective beta3-adrenoceptor agonist, AJ-9677, was reported to ameliorate obesity and insulin resistance in KK-Ay mice. We examined the acute and chronic effects of AJ-9677 on obese dogs. Oral administration of AJ-9677 (0.01 or 0.1 mg/kg) to overnight fasted obese beagles produced a dose-dependent rise in the plasma levels of non-esterified fatty acids and insulin in 1h, followed by a gradual drop of the plasma glucose level. It produced no apparent abnormal behaviors, but easily detectable cutaneous flushing. Daily treatment of AJ-9677 at a lower dose (0.01 mg/kg) for three weeks produced no notable change in body weight, but at a higher dose (0.1 mg/kg) it reduced the body weight compared to a placebo treatment after seven weeks. Computed tomographic examinations revealed a remarkable reduction of body fat after the AJ treatment, being consistent with the histological observations that the adipose tissue of AJ-9677-treated dogs consisted of smaller and some multilocular adipocytes. The plasma levels of leptin and adiponectin were decreased and increased, respectively, after the AJ treatment, reflecting the reduction of adiposity. It was concluded that AJ-9677 is useful for the treatment of obesity in the dog.

The aim of this study was to examine the effect of proinsulin C-peptide on the autonomic nervous systems in rats. Intravenous administration of C-peptide gradually increased electrophysiological activity of the vagus nerves into the stomach and pancreas for at least 90 min. It also slightly increased gastric acid secretion that was suppressed by the treatment with atropine. Intraperitoneal injection of C-peptide did not affect the basal and stress-induced norepinephrine turnover rate, a biochemical index of sympathetic nerve activity. These results indicate that C-peptide increases parasympathetic nerve activity without affecting sympathetic nerve activity. This could explain, at least in part, the ameliorating effects of C-peptide on impaired cardiac autonomic nerve functions in patients with type 1 diabetes., http://www.sciencedirect.com/science/journal/01969781

Numerous studies show protective effects of C-peptide on the development of the diabetic complications in type 1 diabetic patients and in its animal models. Recently, we have demonstrated that streptozotocin-induced diabetic rats with almost complete reduction of endogenous insulin and C-peptide increase expression of renal endothelial nitric oxide synthase (eNOS), and that replacement with C-peptide abrogates the increase of eNOS without affecting hyperglycemic state. Here, we first summarize current topics on nitric oxide (NO)-eNOS system in ­diabetic nephropathy and then we discuss the mechanisms underlying the ameliorating effects of C-peptide on diabetic nephropathy with focus on the NO-eNOS system.

AIMS/HYPOTHESIS: C-peptide reduces renal damage in diabetic patients and experimental animal models. In vitro studies suggest that the renal effects of C-peptide may, in part, be explained by stimulation of Na(+)/K(+)-ATPase activity. However, the responses of Na(+)/K(+)-ATPase expression in the kidney of diabetic animals to C-peptide administration remain unclear. The aim of this study was to clarify the responses.METHODS: Type 1 diabetic rats were produced by injecting streptozotocin (STZ), and some of the rats were treated with either C-peptide or insulin by the aid of an osmotic pump for 1 week. The mRNA expression and immunohistochemical localization of Na(+)/K(+)-ATPase alpha1-, alpha2- and beta3-subunits were investigated in the kidney of these rats.RESULTS: Na(+)/K(+)-ATPase alpha1-subunit was abundantly expressed in the medullary collecting ducts of control animals, but the expression was markedly decreased in the diabetic state with concomitant decrease in its mRNA expression. Similar decreases were observed in the insulin-treated diabetic rats, whereas in the C-peptide-treated diabetic rats, there was no reduction in the alpha1-expression. The beta3-subunit was expressed in podocytes and parietal cells in the glomeruli, vascular endothelial cells, and cortical collecting ducts, but lesser signals were observed in the proximal and distal tubules. However, the beta3-subunit did not appear to be affected by the diabetic state.CONCLUSIONS: Diabetes selectively reduced Na(+)/K(+)-ATPase alpha1-subunit expression and abundance. Chronic administration of C-peptide prevented this decrease. This implies a role for C-peptide in the long-term regulation of Na(+)/K(+)-ATPase function.

To characterize the roles of C-peptide in vascular homeostatic processes, we examined the genes regulated by C-peptide in LEII mouse lung microvascular endothelial cells. Treatment of the cells with C-peptide increased the expression of c-Jun N-terminal kinase 1 (JNK1) mRNA dose-dependently, accompanied by an increase in JNK1 protein content. Prior treatment of the cells with PD98059, an ERK kinase inhibitor or SB203580, a p38MAPK inhibitor, abrogated the C-peptide-elicited JNK1 mRNA expression. These results indicate that C-peptide increases JNK1 protein levels, possibly through ERK- and p38MAPK-dependent activation of JNK gene transcription.

Retinol-binding protein 4 (RBP4) is a plasma protein involved in retinol transportation, and recent evidence in rodents suggests that RBP4 is also a metabolic regulator that modifies insulin sensitivity. To assess how RBP4 levels are regulated in ruminants, we determined the RBP4 concentrations in bovine plasma and milk using Western blot analysis. Plasma RBP4 levels in non-pregnant non-lactating (control) cows were around 45 μg/ml, which were sustained during 60-h fasting, but decreased significantly 4 h after lipopolysaccharide (LPS) administration. Basal plasma retinol concentration was around 30 μg/dl, but this decreased to approximately one-third and one-half of these values during fasting and 8 h after LPS challenge, respectively. Plasma RBP4 and retinol levels in cows 3–6 d before parturition were comparable to those of the controls. However, on the day of parturition both were significantly decreased and had returned to basal levels by two weeks after calving. Interestingly, RBP4 was clearly detected in colostrum (16·4±5·6 μg/ml) but was only faintly detected in milk from cows at 7 d and 15 d after calving. Retinol concentrations in colostrum were almost 10-fold higher than those in plasma, while those in milk were comparable to those in plasma. These results suggest that RBP4 and retinol levels are independently regulated under physiological and pathophysiological conditions and that RBP4, like retinol, is transferred from maternal stores to calves through colostrum.

Previously, we demonstrated that hepatocyte growth factor (HGF) potently stimulates endothelial nitric oxide synthase (eNOS) activity and nitric oxide (NO) production through a calcium- and Akt-mediated phosphorylation at Ser-1179 (Ser-1177 human) in bovine aortic endothelial cells. The regulation of eNOS, however, also involves interaction with chaperone proteins such as heat shock protein (HSP) 90, which can be enhanced by agonist stimulation of the enzyme. In the present work, the role of HSP90 in HGF stimulation of eNOS was examined in an endothelial cell culture system. Treatment of endothelial cells with geldanamycin, a commonly used HSP90 inhibitor, augmented HGF-stimulated eNOS phosphorylation at Ser-1179, while it did not alter eNOS phosphorylation at Thr-497. However, other HSP90 inhibitors, namely 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) and radicicol, did not possess similar effects. Neither HGF nor geldanamycin treatment, independently or in combination, altered HSP90/eNOS interaction in endothelial cells. In addition, geldanamycin treatment did not enhance the HGF-induced phosphorylation of Akt, ERK1/2 and p38MAPK. Src kinase inhibition by PP2 also failed to block the geldanamycin effects. These results suggest that geldanamycin, but neither 17-AAG nor radicicol, may enhance HGF-mediated eNOS Ser-1179 phosphorylation by some as yet unknown mechanisms independently of HSP90 inhibition.

We examined the effect of stroma-derived factors, hepatocyte growth factor (HGF) and leptin, on morphological differentiation of bovine mammary epithelial cells (BMEC) in collagen gel three-dimensional culture in vitro. BMEC treated with HGF, but not leptin, formed duct-like organoids. The formation of organoids by HGF was enhanced by treatment with a mixture of insulin, cortisol and prolactin, while BMEC treated with the mixture alone did not produce the organoid. In contrast, the formation of organoids by HGF was dose-dependently inhibited by simultaneous addition of leptin, regardless of the presence or absence of the hormone mixture. These results suggest that stroma-derived factors intricately regulate mammary epithelial morphogenesis.

application/pdf, Mammary glands are physiologically active in female mammals only during nursing. Immediately after weaning, most lactation-related genes are downregulated and milk production ceases. In our previous study, we have detected an inwardly rectifying potassium channel (Kir) 2.1-like current in mammary secretory (MS) cells freshly isolated from lactating mice. This current is highly sensitive to external Ba2+ . The potassium permeability of the Kir channels may contribute to the secretion and/or preservation of ions in milk. We hypothesized that the functions of the Kir channels in MS cells are regulated after weaning. To test this hypothesis, we examined the effect of forced weaning on the Ba2+ -sensitive Kir current and Kir2.1 expression in the mouse mammary glands. Twenty-four hours after weaning, the lumina of mammary acini were histologically enlarged by milk accumulation. The whole-cell patch-clamp analyses showed that the Ba2+ -sensitive Kir current in the post-weaning MS cells was smaller than in the lactating MS cells. The inward conductances of the current in the lactating and post-weaning cells were 4.25 ± 0.77 and 0.93 ± 0.34 nS, respectively. Furthermore, real-time PCR and Western blot analyses showed that Kir2.1 mRNA and protein expression decreased in the post-weaning mammary gland (mRNA, 90% reduction; protein, 47% reduction). Moreover, the local milk accumulation caused by teat sealing decreased Kir conductance in MS cells (2.74 ± 0.45 and 0.36 ± 0.27 nS for control and sealed mammary glands, respectively). This was concomitant with the reduction in the Kir2.1 mRNA expression. Our results suggest that milk stasis after weaning immediately decreases the Kir conductance in MS cells. This decrease in the Kir conductance may be partly caused by the reduction in the Kir2.1 mRNA and protein expression. These alterations during the post-weaning period may be involved in the cessation of ion secretion and/or preservation in the milk.