Long noncoding RNA HBBP1 enhances γ-globin expression through the ETS transcription factor ELK1.

ß-Thalassemia is an autosomal recessive genetic disease caused by defects in the production of adult hemoglobin (HbA, α2ß2), which leads to an imbalance between α- and non-α-globin chains. Reactivation of γ-globin expression is an effective strategy to treat ß-thalassemia patients. Previously, it was demonstrated that hemoglobin subunit beta pseudogene 1 (HBBP1) is associated with elevated fetal hemoglobin (HbF, α2γ2) in ß-thalassemia patients. However, the mechanism underlying HBBP1-mediated HbF production is unknown. In this study, using bioinformatics analysis, we found that HBBP1 is involved in γ-globin production, and then preliminarily confirmed this finding in K562 cells. When HBBP1 was overexpressed, γ-globin expression was increased at the transcript and protein levels in HUDEP-2 cells. Next, we found that ETS transcription factor ELK1 (ELK1) binds to the HBBP1 proximal promoter and significantly promotes its activity. Moreover, the synthesis of γ-globin was enhanced when ELK1 was overexpressed in HUDEP-2 cells. Surprisingly, ELK1 also directly bound to and activated the γ-globin proximal promoter. Furthermore, we found that HBBP1 and ELK1 can interact with each other in HUDEP-2 cells. Collectively, these findings suggest that HBBP1 can induce γ-globin by enhancing ELK1 expression, providing some clues for γ-globin reactivation in ß-thalassemia.

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-ß (TGF-ß) 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-ß, a key effector of pancreatic pathophysiology. This novel network may be amenable to pharmacologic manipulations during CP in humans.

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

Activation of pancreatic stellate cells (PSCs) by transforming growth factor (TGF)-ß 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-ß superfamily, have anti-fibrogenic functions, in contrast to TGF-ß, 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-ß treatment. BMP2 pretreatment inhibited TGF-ß-induced α-SMA, fibronectin, and collagen type Ia expression. Knocking down Smad1 with small-interfering RNA reversed the inhibitory effect of BMP2 on TGF-ß-induced α-SMA and fibronectin expression. Thus, BMP2 opposes the fibrogenic function of TGF-ß in PSCs through the Smad1 signaling pathway.

Gremlin1 and TGF-ß1 protect kidney tubular epithelial cells from ischemia-reperfusion injury through different pathways.

BACKGROUND: Gremlin1 belongs to the superfamily members of transforming growth factor (TGF)-ß1, playing a profibrotic role in chronic kidney diseases (CKD) and the transition from the late stage of acute kidney injury (AKI) to CKD, but the effect it plays in the early stage of AKI is unclear. This study aimed to investigate the role of Gremlin1on apoptosis in renal tubular epithelial cells under ischemia-reperfusion (I/R) induction. METHODS: We detected Gremlin1 and TGF-ß1 expression in the kidneys of mice undergoing renal ischemia-reperfusion injury bilaterally. We induced apoptosis through depletion and reperfusion of oxygen and serum in human kidney tubular epithelial cells (HKCs), mimicking I/R injury in vivo, and detected the role and pathways of Gremlin1 and TGF-ß1on HKCs injury. RESULTS: Mice undergoing bilateral I/R surgery presented AKI with a significant increase in serum creatinine, obvious renal tubular injuries, and increased macrophage cell and T-cell infiltration in interstitial areas. Gremlin1 expression was significantly increased along with TGF-ß1 in the kidneys of AKI mice compared to sham mice. Exogenous Gremlin1 inhibited I/R-induced caspase3 expression in HKCs, which was blocked by a VEGFR2 kinase inhibitor III (SU5416). TGF-ß1 also inhibited I/R-induced cell apoptosis in HKCs but had no synergic effect with Gremlin1. The TGF-ß1's inhibitory effect could be blocked by the TGF-ß1 type I receptor (activin receptor-like kinase 5, and ALK5)-specific inhibitor SB431542. CONCLUSIONS: Gremlin1 and TGF- ß1 protect kidney tubular epithelial cells from ischemia-reperfusion-induced apoptosis through VEGFR2 and Smad2 signaling pathways.

Reactivation of γ-globin expression using a minicircle DNA system to treat ß-thalassemia.

Reactivation of fetal hemoglobin by editing the B-cell lymphoma/leukemia 11A (BCL11A) erythroid enhancer is an effective gene therapy for ß-thalassemia. Using the CRISPR/Cas9 system, fetal γ-globin expression can be robustly reactivated to mitigate the clinical course of ß-thalassemia. In our study, we found that the transfection efficiencies of CD34+ hematopoietic stem/progenitor cells (HSPCs) were significantly and negatively correlated with the length of plasmids and greatly affected by the linearization of plasmids. Furthermore, the transgene expression of minicircles (MC) without plasmid backbone sequences was better both in vitro and in vivo compared with conventional plasmids. Thus, MC DNA was used to deliver the cassette of Staphylococcus aureus Cas9 (SaCas9) into HSPCs, and a single-guide RNA targeting the erythroid enhancer region of BCL11A was selected. After electroporation with MC DNA, an evident efficiency of gene editing and reactivation of γ-globin expression in erythroblasts derived from unsorted HSPCs was acquired. No significant off-target effects were found by deep sequencing. Furthermore, fragments derived from lentiviral vectors, but not MC DNA, were highly enriched in promoter, exon, intron, distal-intergenic, and cancer-associated genes, indicating that MC DNA provided a relatively safe and efficient vector for delivering transgenes. The developed MC DNA vector provided a potential approach for the delivery of SaCas9 cassette and the reactivation of γ-globin expression for ameliorating syndromes of ß-thalassemia.

Dietary fiber enhances TGF-ß 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-ß signaling in rat intestinal epithelial cells (RIE-1). Furthermore, TGF-ß represses inhibitors of differentiation (Ids), leading to apoptosis. We hypothesized that dietary fiber enhances TGF-ß's 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-ß(1), and cell functions were evaluated. Furthermore, the role of Ids in butyrate- and TGF-ß-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-ß alone inhibited cell growth and induced cell cycle arrest. The combined treatment of butyrate and TGF-ß 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-ß 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.

Connective tissue growth factor stimulates renal cortical myofibroblast-like cell proliferation and matrix protein production.

Myofibroblasts primarily contribute to the pathogenesis of renal interstitial fibrosis by unregulated cell proliferation and synthesis of excessive amounts of extracellular matrix (ECM) proteins. We used cultured myofibroblast-like cells obtained by outgrowth from explants of rat kidney cortex to study the effects and relevant signaling pathway of connective tissue growth factor (CTGF) on cell proliferation and ECM production. Exogenous CTGF stimulated proliferation of myofibroblast-like cells in a dose- and time-dependent manner. CTGF also increased the secretion of fibronectin and collagen I protein in the supernatant medium. Nevertheless, CTGF did not affect matrix-degrading metalloproteinases-2 and -9 activities in supernatant medium measured by gelatin zymography. CTGF induced activation of extracellular signal-regulated protein kinase (ERK)1/2 mitogen-activated protein kinase pathway as early as 5 minutes. Inhibition of ERK1/2 activation with PD98059 completely blocked CTGF-induced cell proliferation as well as secretion of fibronectin and collagen I protein. The above results indicate that CTGF triggers cell proliferation and production of ECM proteins in cultured myofibroblast-like cells through the ERK1/2 mitogen-activated protein kinase pathway.

Transforming Growth Factor-ß 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 µg/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)-ß (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-ß 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-ß and BMP2 may function through inverse regulation of miR-200b levels.

Gremlin is a key pro-fibrogenic factor in chronic pancreatitis.

UNLABELLED: The current study aims to identify the pro-fibrogenic role of Gremlin, an endogenous antagonist of bone morphogenetic proteins (BMPs) in chronic pancreatitis (CP). CP is a highly debilitating disease characterized by progressive pancreatic inflammation and fibrosis that ultimately leads to exocrine and endocrine dysfunction. While transforming growth factor (TGF)-ß is a known key pro-fibrogenic factor in CP, the TGF-ß superfamily member BMPs exert an anti-fibrogenic function in CP as reported by our group recently. To investigate how BMP signaling is regulated in CP by BMP antagonists, the mouse CP model induced by cerulein was used. During CP induction, TGF-ß1 messenger RNA (mRNA) increased 156-fold in 2 weeks, a BMP antagonist Gremlin 1 (Grem1) mRNA levels increased 145-fold at 3 weeks, and increases in Grem1 protein levels correlated with increases in collagen deposition. Increased Grem1 was also observed in human CP pancreata compared to normal. Grem1 knockout in Grem1 (+/-) mice revealed a 33.2 % reduction in pancreatic fibrosis in CP compared to wild-type littermates. In vitro in isolated pancreatic stellate cells, TGF-ß induced Grem1 expression. Addition of the recombinant mouse Grem1 protein blocked BMP2-induced Smad1/5 phosphorylation and abolished BMP2's suppression effects on TGF-ß-induced collagen expression. Evidences presented herein demonstrate that Grem1, induced by TGF-ß, is pro-fibrogenic by antagonizing BMP activity in CP. KEY MESSAGES: • Gremlin is upregulated in human chronic pancreatitis and a mouse CP model in vivo. • Deficiency of Grem1 in mice attenuates pancreatic fibrosis under CP induction in vivo. • TGF-ß induces Gremlin mRNA and protein expression in pancreatic stellate cells in vitro. • Gremlin blocks BMP2 signaling and function in pancreatic stellate cells in vitro. • This study discloses a pro-fibrogenic role of Gremlin by antagonizing BMP activity in chronic pancreatitis.

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-ß 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 pp38(MAPK) were further enhanced in BMPR2(+/-) mice compared to wild-type mice (P<0.05). In vitro, BMP2 inhibited TGF-ß-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-ß 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 p38(MAPK) signaling pathways.

PTHrP is a novel mediator for TGF-ß-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)-ß is an important tumor suppressor that induces cell cycle arrest and apoptosis. Increased PTHrP expression has been implicated in TGF-ß-induced growth inhibition in human hepatocellular carcinoma cells. However, whether PTHrP is involved in TGF-ß-induced apoptosis remains unknown. Using Hep3B and HuH-7, two human hepatocellular carcinoma cell lines, the current study examined the hypothesis that TGF-ß-induced apoptosis is mediated by the induction of PTHrP expression. We found that (1) TGF-ß 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-ß-induced apoptosis; and (3) TGF-ß-induced PTHrP expression is Smad3-dependent. Thus, we have identified PTHrP as a novel mediator for TGF-ß-induced apoptosis in Hep3B cells. Our findings provide further insights into the mechanisms through which TGF-ß conveys tumor suppression activity.

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 µg/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.