You-Qing Zhang

Translocation of a calcium-permeable cation channel induced by insulin-like growth factor-I

Calcium plays a critical part in the regulation of cell growth, and growth factors stimulate calcium entry into cells through calcium-permeable channels. However, the molecular nature and regulation of calcium-permeable channels are still unclear at present. Here we report the molecular characterization of a calcium-permeable cation channel that is regulated by insulin-like growth factor-I (IGF-I). This channel, which we name growth-factor-regulated channel (GRC), belongs to the TRP-channel family and localizes mainly to intracellular pools under basal conditions. Upon stimulation of cells by IGF-I, GRC translocates to the plasma membrane. Thus, IGF-I augments calcium entry through GRC by regulating trafficking of the channel.

IMPAIRED DIFFERENTIATION OF ENDOCRINE AND EXOCRINE CELLS OF THE PANCREAS IN TRANSGENIC MOUSE EXPRESSING THE TRUNCATED TYPE II ACTIVIN RECEPTOR

Activin A is expressed in endocrine precursor cells of the fetal pancreatic anlage. To determine the physiological significance of activins in the pancreas, a transgenic mouse line expressing the truncated type II activin receptor under the control of beta-actin promoter was developed. Histological analyses of the pancreas revealed that the pancreatic islets of the transgenic mouse were small in size and were located mainly along the pancreatic ducts. Immunoreactive insulin was detected in islets, some acinar cells, and in some epithelial cells in the duct. In addition, there were abnormal endocrine cells outside the islets. The shape and the size of the endocrine cells varied and some of them were larger than islets. These cells expressed immunoreactive insulin and glucagon. In the exocrine portion, there were morphologically abnormal exocrine cells, which did not form a typical acinar structure. The cells lacked spatial polarity characteristics of acinar cells but expressed immunoreactive amylase, which was distributed diffusely in the cytoplasm. Plasma glucose concentration was normal in the transgenic mouse before and after the administration of glucose. The insulin content of the pancreas in transgenic and normal mice was nearly identical. These results suggest that activins or related ligands regulate the differentiation of the pancreatic endocrine and exocrine cells.

A Single Intraportal Administration of Follistatin Accelerates Liver Regeneration in Partially Hepatectomized Rats

BACKGROUND/AIMS Activin A is an autocrine negative regulator of DNA synthesis in rat hepatocytes and is expressed in remnant liver after partial hepatectomy. To determine the role of activin A in liver regeneration, the effects of exogenous follistatin, which blocks the action of activin A, were examined. METHODS Human recombinant follistatin was infused into the portal vein immediately after 70% hepatectomy. Changes in body weight, remnant liver weight, liver regeneration rate, and nuclear bromodeoxyuridine labeling were measured. RESULTS In control rats, nuclear labeling was observed at 24 hours and peaked at 36 hours after the hepatectomy. In follistatin-treated rats, nuclear labeling was first observed after 18 hours and was significantly (P < 0.05) greater than that in control rats at 24 hours. In follistatin-treated rats, both remnant liver weight and liver regeneration rate were significantly greater at 120 hours. Serum concentrations of albumin and glucose remained reduced for up to 120 hours in control rats but recovered in follistatin-treated rats. CONCLUSIONS A single administration of follistatin accelerates the initial round of DNA synthesis after partial hepatectomy. Activin A produced in remnant liver may exert tonic inhibitory effect on liver regeneration. Follistatin may be useful as a potential therapeutic agent to promote liver regeneration.

Upregulatory Expression of Furin and Transforming Growth Factor-β by Fluid Shear Stress in Vascular Endothelial Cells

Abstract—Furin, a yeast Kex2-family endoprotease, converts many vasoregulatory propeptides, including pro-transforming growth factor (TGF)-&bgr; to their mature forms. We examined whether furin expression is regulated by shear stress in vivo and in vitro. When an arteriovenous shunt was placed between the carotid artery and external jugular vein in rabbits, furin and TGF-&bgr; were highly expressed in shear stress-loaded endothelial cells. Exposure of bovine aortic endothelial cells in culture to shear stress induced furin and TGF-&bgr; expression in a similar manner. Molecular analysis of furin expression in bovine aortic endothelial cells revealed that shear stress increases the furin gene expression at transcriptional levels. Furthermore, TGF-&bgr; itself increased the furin mRNA levels. Shear-mediated furin expression was partly mediated by TGF-&bgr; because shear-induced furin mRNA levels were considerably decreased by overexpression of the truncated form of the TGF-&bgr; type II receptor. Likewise, blockade of furin activity by a furin inhibitor significantly decreased the endothelial production of mature TGF-&bgr;. Taken together, the results indicate that furin expression is induced and maintained by a coordination of shear stress and TGF-&bgr;. Increased furin expression may facilitate the formation of mature TGF-&bgr;, resulting in the enhanced effects of TGF-&bgr; on endothelial cells and vascular smooth muscle cells in the vasculature.

Immediate onset of DNA synthesis in remnant rat liver after 90% hepatectomy by an administration of follistatin

BACKGROUND/AIM Follistatin is an antagonist of activins and is effective in promoting liver regeneration after 70% hepatectomy. To examine its efficacy under more critical conditions, we studied the effect of follistatin on liver regeneration in 90% hepatectomized rat. METHODS Human recombinant follistatin was infused into the portal vein immediately after 90%, hepatectomy in 24-h-starved rats, and changes in the liver regeneration rate and nuclear bromodeoxyuridine labeling were measured. RESULTS In control rats, nuclear labeling was first detected at 11 h of hepatectomy. In follistatin-treated rats, nuclear labeling was markedly increased at 3 h, and was significantly higher than that in control rats at 24, 72, 96, 120 and 144 h. The liver regeneration rate was significantly higher in follistatin-treated rats at 48, 72, 96, 120, 144 and 168 h. To determine the reason for the accelerated growth in starved rats, we compared the expression of mRNA for c-myc, p53, p21CIP1, p15INK4B, p27KIP1, and subunits of activins in fed and starved rats. mRNA for p21CIP1 and p15INK4B, but not p27KIP1 were decreased in 24 h-starved rats compared to the fed rats. mRNA for betaA subunit of activin was not detected in either fed or 24-h-starved rats, whereas that for betaC subunit was increased in starved rats. CONCLUSION These results indicate that follistatin induces immediate onset of DNA synthesis in 90% hepatectomized rats and is quite effective in promoting liver regeneration.

REGULATION OF THE EXPRESSION OF FOLLISTATIN IN RAT HEPATOCYTES

To elucidate the regulation of follistatin production in the liver, we studied changes in steady-state follistatin mRNA levels in cultured rat hepatocytes. Activin A stimulated follistatin mRNA levels in a time- and concentration-dependent manner. The stimulatory effect of activin A on follistatin mRNA was significant at 2 h, maximal at 6 h and declined thereafter. Incubating the cells with EGF increased follistatin mRNA levels at 48 h and later. The EGF-induced increase in follistatin mRNA was markedly inhibited by exogenous follistatin in the culture medium, which blocks the action of activin A synthesized in hepatocytes, suggesting that endogenous activin A at least partly mediated the effect of EGF. We also examined the effects of transforming growth factor-beta (TGF-beta), glucagon and alpha-adrenergic agonist, phenylephrine, on follistatin mRNA levels. TGF-beta increased the follistatin mRNA to levels similar to those caused by activin A. Phenylephrine and glucagon also increased follistatin mRNA levels but the effects were transient and weaker than those caused by activin A. Finally, follistatin mRNA levels were markedly increased in remnant liver 3 h after 70% hepatectomy. The mRNA remained elevated for up to 72 h. These results indicate that the expression of mRNA for follistatin is positively controlled by activin A, TGF-beta and other hormones or neurotransmitters. The stimulatory effect of EGF on follistatin mRNA is mediated by activin A released from hepatocytes.

Involvement of Smad proteins in the differentiation of pancreatic AR42J cells induced by activin A

Aims/hypothesis. Activin A induces differentiation of amylase-secreting pancreatic AR42J cells into endocrine cells. This study assesses the role of Smad proteins in the actions of activin A in AR42J cells. Methods. The expression of Smad proteins was determined by northern blotting. Phosphorylation and translocation of Smad2 was measured by transfecting flag-tagged Smad2. Involvement of Smad2 was examined by transfecting cDNA encoding N-terminal and C-terminal domains of Smad2. Results. The mRNAs for Smad2 and Smad4 were abundantly expressed whereas the expression of mRNA for Smad1 and Smad3 was very low. Activin A induced serine-phosphorylation and the subsequent accumulation of the Smad2 in nuclei. Transfection of the N-terminal domain of Smad2, which acts as a dominantly negative mutant (Smad2-N), blocked the morphological changes induced by activin A whereas the C-terminal domain of Smad2, which acts as a constitutively active mutant (Smad2-C), reproduced the activin-induced morphological changes. Similarly, Smad2-N blocked apoptosis induced by activin A and Smad2-C induced apoptosis. Activin A converted AR42J into insulin-secreting cells in the presence of hepatocyte growth factor and introduction of Smad2-N inhibited the differentiation. Smad2-C, however, did not induce differentiation in the presence of hepatocyte growth factor. Conclusions/interpretation. Activation of the Smad2 pathway is necessary and sufficient to induce apoptosis and morphological changes. Although activation of the Smad2 pathway is required, it is not solely sufficient for the differentiation of AR42J into endocrine cells. [Diabetologia (1999) 42: 719–727]

Derangements in the activin-follistatin system in hepatoma cells☆

BACKGROUND/AIMS The growth of normal hepatocytes is regulated by the activin-follistatin system. The aim of this study was to investigate the activin-follistatin system in hepatoma cells. METHODS The production and action of activin and follistatin in human hepatoma cell lines were examined. Activin A and follistatin were measured by bioassay and protein-binding assay, respectively. RESULTS Activin A inhibited cell growth in HepG2 cells but not in either PLC/PRF/5 or HLE cells. However, the effect of activin A in HepG2 cells was attenuated at high cell density. In HepG2 cells, two classes of activin-binding sites were expressed, and affinity cross-linking showed that 125I-activin A bound specifically to three proteins with molecular weights of 48, 67, and 94 kilodaltons. In PLC/PRF/5 cells, a single class of binding site was observed, and the binding capacity was approximately 60% of the capacity in HepG2 cells. Virtually no 125I-activin A binding was detected in HLE cells. Bioactivity and messenger RNA for activin A were undetectable in three cell lines. In contrast, follistatin was released from three cell lines. CONCLUSIONS Multiple alterations in the activin-follistatin system were found in three hepatoma cell lines. The accelerated growth observed in hepatoma cells may be caused, at least partly, by the attenuation of the action of activin A.

Role of mitogen-activated protein kinase and phosphoinositide 3-kinase in the differentiation of rat pancreatic AR42J cells induced by hepatocyte growth factor

Aims/hypothesis. Pancreatic AR42J cells express both exocrine and neuroendocrine properties. When exposed to activin A, approximately 50 % of the cells die within 3 days by apoptosis. Addition of hepatocyte growth factor prevents apoptosis induced by activin A and induces differentiation into insulin-producing cells. The present study was conducted to examine the role of mitogen-activated protein kinase and phosphoinositide 3-kinase in the action of hepatocyte growth factor. Methods. The role of mitogen-activated protein kinase was assessed by using 2-(2 ′-amino-3 ′-methoxyphenol)-oxanaphthalen-4-one (PD098 059). Cells were also transfected with cDNA for mitogen-activated protein kinase phosphatase and constitutively active mutant of mitogen-activated protein kinase kinase. Results. Hepatocyte growth factor induced sustained activation of the mitogen-activated protein kinase, which was inhibited by PD098 059. PD098 059 completely blocked the differentiation and also blocked the prevention of apoptosis. Transfection of the cells with cDNA for mitogen-activated protein kinase phosphatase reproduced the effect of PD098 059. Conversely, transfection with cDNA for the constitutively active mutant of mitogen-activated protein kinase kinase reproduced the effect of hepatocyte growth factor. In contrast, addition of wortmannin or transfection of the dominantly negative form of the p85 subunit of the phosphoinositide 3-kinase did not affect differentiation induced by hepatocyte growth factor. Instead, wortmannin enhanced the increase in the insulin content of the differentiated AR42J cells. Conclusion/interpretation. The MAP kinase pathway is necessary and sufficient for the action of HGF on differentiation of AR42J cells. [Diabetologia (1999) 42: 450–456]

Role of the activin-follistatin system in the morphogenesis and regeneration of the renal tubules.

Activin A inhibits branching tubulogenesis of the kidney during development. Activin A also inhibits branching tubulogenesis in MDCK cells, an in vitro tubulogenesis model. On the other hand, follistatin, an antagonist of activin A, reverses the effect of activin A and induces branching tubulogenesis. Follistatin also promotes tubular regeneration after ischemia/reperfusion injury. The activin/follistatin system is one of the important regulatory systems modulating developmental and regeneration processes of the kidney.