Chordin-like 2 influences the differentiation fate of retinal pigment epithelium cells by dynamically regulating BMP pathway

AIM:To explore the functions of Chordin-like 2,which is encoded by CHRDL2,in the process of retinal pigmented epithelium(RPE)differentiation and damage repair.METHODS:The fetal RPE cells(f RPE)was obtained from aborted fetus which obeyed medical ethics.Real-time quantitative polymerase chain reaction was used to measure expression quantity of CHRDL2 and other functional genes expression.Knocking down and overexpression was used to analyze the functions about Chordin-like 2.Enzyme-linked immunosorbent assay(ELISA)was used to detect the secretion of bone morphogenetic proteins 4(BMP4).Flow cytometry was used to analyze cell cycle.Cell morphology was observed by phase contrast microscope(PCM).RESULTS:In normal RPE cells,CHRDL2 was firstly upregulated and followed a downregulation but eventually,it was expressed higher than the cells which undergone epithelial-mesenchymal transition(EMT).After knocking down CHRDL2,the secretion of BMP4 was decreased,RPErelated genes(OTX2,MITF,RPE65)were downregulated while EMT-related genes(SNAI1,VIM)were upregulated.However,the expression of these related genes after overexpression of CHRDL2 had contrary results.Chordin-like 2 also regulated the cell cycle by regulating BMP pathway.When CHRDL2 was knocked down,more f RPE cells stayed in S phase of cell cycle,while adding BMP4 reduced the proportion of the cells in S phase.However,overexpression of CHRDL2 increased more BMP4 secretion,this effect decreased the number of cells in S phase,but exogenous BMP inhibitor also could change this effect.At last,in the process of RPE cells differentiation,adding BMP4 at early stage could intervene normal RPE differentiation.Compared with BMP4,inhibiting BMP pathway had no significant negative effect at early stage,but suppressed differentiation at late stage.CONCLUSION:BMP pathway can be activated in a correct temporal order,otherwise,the cells have incorrect differentiation orientation.And Chordin-like 2 plays a role in dynamic regulation of BMP pathway and it also regulates the differentiation of RPE cells.Therefore,this research enlightens a new direction to inhibit EMT and promote cell redifferentiation after injury.

Nystose attenuates bone loss and promotes BMSCs differentiation to osteoblasts through BMP and Wnt/β-catenin pathway in ovariectomized mice

Increasing the osteogenic differentiation ability and decreasing the adipogenic differentiation ability of bone marrow mesenchymal stem cells(BMSCs)is a potential strategy for the treatment of osteoporosis(OP).Naturally derived oligosaccharides have shown significant anti-osteoporotic effects.Nystose(NST),an oligosaccharide,was isolated from the roots of Morinda officinalis How.(MO).The aim of the present study was to investigate the effects of NST on bone loss in ovariectomized mice,and explore the underlying mechanism of NST in promoting differentiation of BMSCs to osteoblasts.Administration of NST(40,80 and 160 mg/kg)and the positive control of estradiol valerate(0.2 mg/kg)for 8 weeks significantly prevented bone loss induced by ovariectomy(OVX),increased the bone mass density(BMD),improved the bone microarchitecture and reduced urine calcium and deoxypyridinoline(DPD)in ovariectomized mice,while inhibited the increase of body weight without significantly affecting the uterus weight.Furthermore,we found that NST increased osteogenic differentiation,inhibited adipogenic differentiation of BMSCs in vitro,and upregulated the expression of the key proteins of BMP and Wnt/β-catenin pathways.In addition,Noggin and Dickkopf-related protein-1(DKK-1)reversed the effect of NST on osteogenic differentiation and expression of the key proteins in BMP and Wnt/β-catenin pathway.The luciferase activities and the molecular docking analysis further supported the mechanism of NST.In conclusion,these results indicating that NST can be clinically used as a potential alternative medicine for the prevention and treatment of postmenopausal osteoporosis.

Deamidation enables pathogenic SMAD6 variants to activate the BMP signaling pathway

The BMP signaling pathway plays a crucial role in regulating early embryonic development and tissue homeostasis.SMAD6 encodes a negative regulator of BMP,and rare variants of SMAD6 are recurrently found in individuals with birth defects.However,we observed that a subset of rare pathogenic variants of SMAD6 consistently exhibited positive regulatory effects instead of the initial negative effects on the BMP signaling pathway.We sought to determine whether these SMAD6 variants have common pathogenic mechanisms.Here,we showed that pathogenic SMAD6 variants accompanying this functional reversal exhibit similar increases in deamidation.Mechanistically,increased deamidation of SMAD6 variants promotes the accumulation of the BMP receptor BMPR1A and the formation of new complexes,both of which lead to BMP signaling pathway activation.Specifically,two residues,N262 and N404,in SMAD6 were identified as the crucial sites of deamidation,which was catalyzed primarily by glutamine-fructose-6-phosphate transaminase 2(GFPT2).Additionally,treatment of cells harboring SMAD6 variants with a deamidase inhibitor restored the inhibitory effect of SMAD6 on the BMP signaling pathway.Conversely,when wild-type SMAD6 was manually simulated to mimic the deamidated state,the reversed function of activating BMP signaling was reproduced.Taken together,these findings show that deamidation of SMAD6 plays a crucial role in the functional reversal of BMP signaling activity,which can be induced by a subset of various SMAD6 variants.Our study reveals a common pathogenic mechanism shared by these variants and provides a potential strategy for preventing birth defects through deamidation regulation,which might prevent the off-target effects of gene editing.

Calycosin-7-O-β-D-glucopyranoside stimulates osteoblast differentiation through regulating the BMP/WNT signaling pathways

The iso fl avone calycosin-7-O-β-D-glucopyranoside(CG) is a principal constituent of Astragalus membranaceus(AR) and has been reported to inhibit osteoclast development in vitro and bone loss in vivo. The aim of this study was to investigate the osteogenic effects of CG and its underlying mechanism in ST2 cells. The results show that exposure of cells to CG in osteogenic differentiation medium increases ALP activity, osteocalcin(Ocal) m RNA expression and the osteoblastic mineralization process. Mechanistically, CG treatment increased the expression of bone morphogenetic protein 2(BMP-2), p-Smad 1/5/8, β-catenin and Runx2, all of which are regulators of the BMP- or wingless-type MMTV integration site family(WNT)/β-catenin-signaling pathways. Moreover, the osteogenic effects of CG were inhibited by Noggin and DKK-1 which are classical inhibitors of the BMP and WNT/β-catenin-signaling pathways, respectively. Taken together, the results indicate that CG promotes the osteoblastic differentiation of ST2 cells through regulating the BMP/WNT signaling pathways. On this basis, CG may be a useful lead compound for improving the treatment of bone-decreasing diseases and enhancing bone regeneration.

Serum Iron Overload Activates the SMAD Pathway and Hepcidin Expression of Hepatocytes via SMURF1

Background and Aims:Liver iron overload can induce hepatic expression of bone morphogenic protein(BMP)6 and activate the BMP/SMAD pathway.However,serum iron overload can also activate SMAD but does not induce BMP6 expression.Therefore,the mechanisms through which serum iron overload activates the BMP/SMAD pathway remain unclear.This study aimed to clarify the role of SMURF1 in serum iron overload and the BMP/SMAD pathway.Methods:A cell model of serum iron overload was established by treating hepatocytes with 2 mg/mL of holo-transferrin(Holo-Tf).A serum iron overload mouse model and a liver iron overload mouse model were established by intraperitoneally injecting 10 mg of Holo-Tf into C57BL/6 mice and administering a high-iron diet for 1 week followed by a low-iron diet for 2 days.Western blotting and real-time PCR were performed to evaluate the activation of the BMP/SMAD pathway and the expression of hepcidin.Results:Holo-Tf augmented the sensitivity and responsiveness of hepatocytes to BMP6.The E3 ubiquitin-protein ligase SMURF1 mediated Holo-Tf-induced SMAD1/5 activation and hepcidin expression;specifically,SMURF1 expression dramatically decreased when the serum iron concentration was increased.Additionally,the expression of SMURF1 substrates,which are important molecules involved in the transduction of BMP/SMAD signaling,was significantly upregulated.Furthermore,in vivo analyses confirmed that SMURF1 specifically regulated the BMP/SMAD pathway during serum iron overload.Conclusions:SMURF1 can specifically regulate the BMP/SMAD pathway by augmenting the responsiveness of hepatocytes to BMPs during serum iron overload.

The role of Smad6 in immunity of the pearl oyster Pinctada fucata martensii

Inhibitory Smads(I-Smads),which belong to the Smad family and inhibit bone morphogenic protein 2(BMP2)signaling by a variety of mechanisms,can suppress innate immunity responses in vertebrates.However,there are no reports for the role of Smad6 in immunity in mollusks.In this study,we showed that Smad6 of the pearl oyster Pinctada fucata martensii was located in the Smad6 cluster of the phylogenetic tree;mRNA expression of Smad6 and Smad3 was up-regulated after lipopolysaccharide and polyinosinic:polycytidylic challenge;and transcript levels of Smad6 and Smad3 showed opposite patterns during wound healing.Under salinity stress,water inflow and outflow in the gills appear to be regulated by BMP2-Smads signals,and BMP2-Smads signaling may be closely related to the immune response.Our results indicate that Smad6 is involved in immunity,that it plays a positive role in the response to immune challenge and an inhibitory role during wound healing,and that Smad6 and Smad3 may work against each other.