Xuxia Gao

BMP2 inhibits TGF-β-induced pancreatic stellate cell activation and extracellular matrix formation

Activation of pancreatic stellate cells (PSCs) by transforming growth factor (TGF)-β is the key step in the development of pancreatic fibrosis, a common pathological feature of chronic pancreatitis (CP). Bone morphogenetic proteins (BMPs), members of the TGF-β superfamily, have anti-fibrogenic functions, in contrast to TGF-β, in the kidney, lung, and liver. However, it is not known whether BMPs have an anti-fibrogenic role in the pancreas. The current study was designed to investigate the potential anti-fibrogenic role of BMPs in the pancreas using an in vivo CP model and an in vitro PSC model. CP was induced by repetitive intraperitoneal injections of cerulein in adult Swiss Webster mice. The control mice received saline injections. Compared with the control, cerulein injections induced a time-dependent increase in acinar injury and progression of fibrosis and a steady increase in inflammation. Cerulein injections also induced increases of the extracellular matrix (ECM) protein fibronectin and of α-smooth muscle actin (α-SMA)-positive stellate cells (PSCs). The mice receiving cerulein injections showed increased BMP2 protein levels and phosphorylated Smad1 levels up to 4 wk and then declined at 8 wk to similar levels as the control. In vitro, the isolated mouse and human PSCs were cultured and pretreated with BMP2 followed by TGF-β treatment. BMP2 pretreatment inhibited TGF-β-induced α-SMA, fibronectin, and collagen type Ia expression. Knocking down Smad1 with small-interfering RNA reversed the inhibitory effect of BMP2 on TGF-β-induced α-SMA and fibronectin expression. Thus, BMP2 opposes the fibrogenic function of TGF-β in PSCs through the Smad1 signaling pathway.

DIETARY FIBER ENHANCES TGF-β SIGNALING AND GROWTH INHIBITION IN THE GUT

Dietary fiber intake links to decreased risk of colorectal cancers. The underlying mechanisms remain unclear. Recently, we found that butyrate, a short-chain fatty acid produced in gut by bacterial fermentation of dietary fiber, enhances TGF-β signaling in rat intestinal epithelial cells (RIE-1). Furthermore, TGF-β represses inhibitors of differentiation (Ids), leading to apoptosis. We hypothesized that dietary fiber enhances TGF-βs growth inhibitory effects on gut epithelium via inhibition of Id2. In this study, Balb/c and DBA/2N mice were fed with a regular rodent chow or supplemented with a dietary fiber (20% pectin) and Smad3 level in gut epithelium was measured. In vitro, RIE-1 cells were treated with butyrate and TGF-β(1), and cell functions were evaluated. Furthermore, the role of Ids in butyrate- and TGF-β-induced growth inhibition was investigated. We found that pectin feeding increased Smad3 protein levels in the jejunum (1.47 ± 0.26-fold, P = 0.045, in Balb/c mice; 1.49 ± 0.19-fold, P = 0.016, in DBA/2N mice), and phospho-Smad3 levels (1.92 ± 0.27-fold, P = 0.009, in Balb/c mice; 1.83 ± 0.28-fold, P = 0.022, in DBA/2N mice). Butyrate or TGF-β alone inhibited cell growth and induced cell cycle arrest. The combined treatment of butyrate and TGF-β synergistically induced cell cycle arrest and apoptosis in RIE-1 cells and repressed Id2 and Id3 levels. Furthermore, knockdown of Id2 gene expression by use of small interfering RNA caused cell cycle arrest and apoptosis. We conclude that dietary fiber pectin enhanced Smad3 expression and activation in the gut. Butyrate and TGF-β induced cell cycle arrest and apoptosis, which may be mediated by repression of Id2. Our results implicate a novel mechanism of dietary fiber in reducing the risk of colorectal cancer development.

Bone morphogenetic protein signaling protects against cerulein-induced pancreatic fibrosis.

Bone morphogenetic proteins (BMPs) have an anti-fibrogenic function in the kidney, lung, and liver. However, their role in chronic pancreatitis (CP) is unknown. The aim of this study was to define the anti-fibrogenic role of BMP signaling in the pancreas in vivo under CP induction. Mice with a deletion of BMP type II receptor (BMPR2+/−) were used in this study in comparison with wild-type mice. CP was induced by repetitive cerulein injection intraperitoneally for 4 weeks, and the severity of CP was evaluated. Pancreatic stellate cells (PSCs) were isolated from the mice and treated with BMP2 and TGF-β in vitro, and extracellular matrix protein (ECM) production was measured. Smad and mitogen-activated protein kinase (MAPK) signaling was also evaluated. BMPR2+/− mice revealed a greater pancreatic fibrosis, PSC activation and leukocyte infiltration after CP induction compared to wild-type mice (P<0.05). Under CP induction, phospho (p)Smad1/5/8 was elevated in wild-type mice and this effect was abolished in BMPR2+/− mice; pSmad2 and pp38MAPK were further enhanced in BMPR2+/− mice compared to wild-type mice (P<0.05). In vitro, BMP2 inhibited TGF-β-induced ECM protein fibronectin production in wild-type PSCs; this effect was abolished in BMPR2+/− PSCs (P<0.05). In BMPR2+/− PSCs, pSmad1/5/8 level was barely detectable upon BMP2 stimulation, while pSmad2 level was further enhanced by TGF-β stimulation, compared to wild-type PSCs (P<0.05). BMPR2/Smad1/5/8 signaling plays a protective role against cerulein-induced pancreatic fibrosis by inhibiting Smad2 and p38MAPK signaling pathways.

Noggin attenuates cerulein-induced acute pancreatitis and impaired autophagy

Objectives To investigate the role of bone morphogenetic protein (BMP) signaling in acute pancreatitis (AP) by administration of noggin, an endogenous BMP antagonist, in a cerulein-induced AP model. Methods Acute pancreatitis was induced by 9 hourly intraperitoneal injections of cerulein (50 &mgr;g/kg). Control mice received phosphate-buffered saline injections. In a separate group, noggin (0.5 mg/kg) was given intraperitoneally at 1 hour before and 2, 4, and 6 hours after AP induction. The mice were euthanized at 1 hour after completion of AP induction. The blood samples and the pancreas were harvested for analysis. Isolated pancreatic acini from normal mice and AR42J cells were treated with BMP2 and cerulein. AR42J cells were also treated with noggin. Phosphorylation of Smad1/5/8 was measured. Results Bone morphogenetic protein signaling was up-regulated in AP mouse pancreas. Bone morphogenetic protein 2 and cerulein-induced phosphorylation of Smad1/5/8 in the acinar cells in vitro, which was blocked by noggin. Noggin administration in vivo attenuated AP induction, decreased vacuole formation in acinar cells, blocked LC3-II levels, and partially restored Beclin-1 and lysosomal-associated membrane protein 2 levels. Conclusions Bone morphogenetic protein signaling seems to promote AP induction and autophagy, as suggested by our study showing that noggin ameliorates AP and partially restores autophagic homeostasis.

PTHrP is a novel mediator for TGF-β-induced apoptosis

Parathyroid hormone-related protein (PTHrP) is a polyhormone secretory protein that plays fundamental roles in the development and function of various tissues. Transforming growth factor (TGF)-β is an important tumor suppressor that induces cell cycle arrest and apoptosis. Increased PTHrP expression has been implicated in TGF-β-induced growth inhibition in human hepatocellular carcinoma cells. However, whether PTHrP is involved in TGF-β-induced apoptosis remains unknown. Using Hep3B and HuH-7, two human hepatocellular carcinoma cell lines, the current study examined the hypothesis that TGF-β-induced apoptosis is mediated by the induction of PTHrP expression. We found that (1) TGF-β induces PTHrP mRNA expression, protein expression and secretion in a time-dependent fashion; (2) knockdown of PTHrP gene expression or neutralization of secreted PTHrP isoforms blocks TGF-β-induced apoptosis; and (3) TGF-β-induced PTHrP expression is Smad3-dependent. Thus, we have identified PTHrP as a novel mediator for TGF-β-induced apoptosis in Hep3B cells. Our findings provide further insights into the mechanisms through which TGF-β conveys tumor suppression activity.

Transforming Growth Factor-β and Bone Morphogenetic Protein 2 Regulation of MicroRNA-200 Family in Chronic Pancreatitis

Objectives To investigate regulation of microRNA (miR)-200 family (a, b, c, 141, and 429) in chronic pancreatitis (CP). This was accomplished by examining miR-200 family levels in a mouse model in vivo and their regulation in pancreatic cells in vitro. Methods Chronic pancreatitis was induced by cerulein for 4 weeks (50 &mgr;g/kg, 5 hourly intraperitoneal injections/day, and 3 days/week). Control mice received normal saline. The pancreata were harvested for fibrosis assessment by Sirius red staining and for miRNA, collagen, and fibronectin levels by quantitative PCR. In vitro, human primary pancreatic stellate cells and human primary pancreatic fibroblast (hPFBs), and rat pancreatic epithelial AR42J cells were treated with vehicle, transforming growth factor (TGF)-&bgr; (1 ng/mL), or BMP2 (50 ng/mL) for 24 hours and then harvested for miRNA analysis. Results In CP, miR-200s were decreased by 56% to 70% and inversely correlated with pancreatic fibrosis, miR-21, and miR-31 (P < 0.05). In vitro, TGF-&bgr; inhibited miR-200b in AR42J cells by 62%, whereas BMP2 increased miR-200b in all 3 cell types in a range of 1.5- to 3.4-fold and inhibited miR-21 in hPFBs by 21% (P < 0.05). Conclusions Both in vivo and in vitro studies suggest an antifibrogenic function of miR-200s in CP. The TGF-&bgr; and BMP2 may function through inverse regulation of miR-200b levels.

Tu1902 Characterization of Inducible BMPR2 Homozygous Knockout Mice

Background. Bone morphogenetic proteins (BMPs), members of transforming growth factor (TGF)-β superfamily, play crucial roles in cellular functions and extracellular matrix production. BMPs transduce their signals upon binding to two different types of serine/threonine kinase receptors, type I (BMPR1) and type II (BMPR2). Studies have shown that heterozygous (BMPR2+/-) mice are phenotypically normal, whereas homozygous (BMPR2-/-) mice die at embryonic stage, thus limiting investigations on BMPR2 biological functions in certain organs and models. This study aims at developing inducible homozygous BMPR2 knockout mice to facilitate studies of BMP function in vivo.Methods. Using Cre-loxP technology, BMPR2flox/ flox mice were bred with Cre-ERT2 to generate inducible homozygous (Cre-BMPR2flox/flox) mice. These mice are alive and phenotypically normal. BMPR2 gene expression is anticipated to be abolished in these mice upon induction of BMPR2 gene deletion. Cre-BMPR2flox/flox knockout (KO) mice (n=5) were intraperitoneally injected with Tamoxifen (TAM) (75 mg/ kg body weight) for 5 days to induce BMPR2 gene deletion. Cre-BMPR2flox/flox (CON) mice (n=4) received corn oil as control. Seven days after completion of injections, the mice were euthanized, and the pancreas and liver tissue were harvested. For comparison of BMPR2 levels, the pancreas and liver samples were also collected from heterozygous (BMPR2+/-, n= 4) and wild-type (WT, BMPR2+/+, n=4) mice. Total RNAs were prepared for quantitative real-time PCR for detection of BMPR2 mRNA, and protein lysates were prepared for Western blotting analysis for detection of BMPR2 protein. Results. As demonstrated in Table 1, BMPR2 mRNA levels reduced in the pancreas in BMPR2+/KO mice compared to WT mice, and in the pancreas and liver in Cre-BMPR2flox/flox KO mice compared to Cre-BMPR2flox/ flox CON mice (p<0.05). BMPR2 protein levels with the sizes of 70-150 kD reduced in the pancreas and liver in BMPR2+/KO mice compared to WT mice, and in the liver in CreBMPR2flox/flox KO mice compared to Cre-BMPR2flox/flox CON mice. Conclusions. BMPR2 gene deletion in BMPR2+/mice results in 52% reduction of BMPR2 protein levels in the pancreas and liver as expected, with a less pronounced reduction of BMPR2 mRNA levels. Compared to BMPR2+/KO mice, BMPR2 mRNA and protein levels in Cre-BMPR2flox/flox KOmice reduced more pronounced in the liver, but not in the pancreas. Since Cre-BMPR2flox/ flox mice survive developmental stage, therefore, the adult mice may be a useful tool for studies of BMP functions in the liver. Table 1. BMPR2 mRNA and protein levels in BMPR2 KO mice

Induction of chronic pancreatitis by pancreatic duct ligation activates BMP2, apelin, and PTHrP expression in mice

Chronic pancreatitis (CP) is a devastating disease with no treatments. Experimental models have been developed to reproduce the parenchyma and inflammatory responses typical of human CP. For the present study, one objective was to assess and compare the effects of pancreatic duct ligation (PDL) to those of repetitive cerulein (Cer)-induced CP in mice on pancreatic production of bone morphogenetic protein-2 (BMP2), apelin, and parathyroid hormone-related protein (PTHrP). A second objective was to determine the extent of cross talk among pancreatic BMP2, apelin, and PTHrP signaling systems. We focused on BMP2, apelin, and PTHrP since these factors regulate the inflammation-fibrosis cascade during pancreatitis. Findings showed that PDL- and Cer-induced CP resulted in significant elevations in expression and peptide/protein levels of pancreatic BMP2, apelin, and PTHrP. In vivo mouse and in vitro pancreatic cell culture experiments demonstrated that BMP2 stimulated pancreatic apelin expression whereas apelin expression was inhibited by PTHrP exposure. Apelin or BMP2 exposure inhibited PTHrP expression, and PTHrP stimulated upregulation of gremlin, an endogenous inhibitor of BMP2 activity. Transforming growth factor-β (TGF-β) stimulated PTHrP expression. Together, findings demonstrated that PDL- and Cer-induced CP resulted in increased production of the pancreatic BMP2, apelin, and PTHrP signaling systems and that significant cross talk occurred among pancreatic BMP2, apelin, and PTHrP. These results together with previous findings imply that these factors interact via a pancreatic network to regulate the inflammation-fibrosis cascade during CP. More importantly, this network communicated with TGF-β, a key effector of pancreatic pathophysiology. This novel network may be amenable to pharmacologic manipulations during CP in humans.