Role of Smad3, acting independently of transforming growth factor‐β, in the early induction of Wnt‐β‐catenin signaling by parathyroid hormone in mouse osteoblastic cells

Parathyroid hormone (PTH) exerts an anabolic action on bone but the mechanisms are incompletely understood. We showed previously that PTH interacts with the canonical Wnt‐β‐catenin signaling pathway via the transforming growth factor (TGF)‐β signaling molecule, Smad3, to modulate osteoblast differentiation and apoptosis. Here, we examined which actions of Smad3 are TGF‐β‐independent in stimulating the osteoblast phenotype and PTH‐induced Wnt‐β‐catenin signaling. For this, the TGF‐β receptor type 1 [activin receptor‐like kinase (ALK5)] inhibitor (SB431542), and a Smad3 mutant in which the site normally phosphorylated by ALK5 is mutated from SSVS to AAVA, was used. PTH induced total β‐catenin and reduced phosphorylated β‐catenin levels at 1, 6, and 24 h in mouse osteoblastic MC3T3‐E1 cells. Transient transfection of Smad3AAVA inhibited the PTH induction of total β‐catenin and reduction of phosphorylated β‐catenin levels at 6 and 24 h, but not at 1 h, indicating that the early effects occur independently of TGF‐β receptor signaling. On the other hand, MC3T3‐E1 cell clones in which Smad3AAVA was stably expressed demonstrated elevated β‐catenin levels, although alkaline phosphatase (ALP) activity and mineralization were unaltered. In contrast, MC3T3‐E1 cell clones in which wild‐type Smad3 was stably expressed exhibited increased ALP activity and mineralization that were decreased by the ALK5 inhibitor, SB431542, although the β‐catenin levels induced in these cells were not modulated. In conclusion, the present study indicates that PTH induces osteoblast β‐catenin levels via Smad3 independently of, and dependently on, TGF‐β in the early and later induction phases, respectively. J. Cell. Biochem. 108: 285–294, 2009. © 2009 Wiley‐Liss, Inc.     

BMP‐2 modulates β‐catenin signaling through stimulation of Lrp5 expression and inhibition of β‐TrCP expression in osteoblasts

Canonical BMP and Wnt signaling pathways play critical roles in regulation of osteoblast function and bone formation. Recent studies demonstrate that BMP‐2 acts synergistically with β‐catenin to promote osteoblast differentiation. To determine the molecular mechanisms of the signaling cross‐talk between canonical BMP and Wnt signaling pathways, we have used primary osteoblasts and osteoblast precursor cell lines 2T3 and MC3T3‐E1 cells to investigate the effect of BMP‐2 on β‐catenin signaling. We found that BMP‐2 stimulates Lrp5 expression and inhibits the expression of β‐TrCP, the F‐box E3 ligase responsible for β‐catenin degradation and subsequently increases β‐catenin protein levels in osteoblasts. In vitro deletion of the β‐catenin gene inhibits osteoblast proliferation and alters osteoblast differentiation and reduces the responsiveness of osteoblasts to the BMP‐2 treatment. These findings suggest that BMP‐2 may regulate osteoblast function in part through modulation of the β‐catenin signaling. J. Cell. Biochem. 108: 896–905, 2009. © 2009 Wiley‐Liss, Inc.     

A TGF‐β‐induced gene, βig‐h3, is crucial for the apoptotic disappearance of the medial edge epithelium in palate fusion

TGF‐β3, TβR‐I, and TGF‐β‐activated Smad2 has been suggested to be a series of signaling molecules for secondary palate fusion. In this article, we show that a gene induced by TGF‐β, βig‐h3, is coincidentally expressed with TGF‐β3 in medial edge epithelial (MEE) cells undergoing apoptosis during normal palatal fusion. βig‐h3 was also highly expressed in the areas of post‐weaning mammary gland cells and developing phalangeal joints in which TGF‐β3 or BMP‐4‐induced apoptosis occurs, respectively. Blocking of βig‐h3 expression in E12.5 embryos with antisense oligodeoxynucleotides (ODN) resulted in cleft of the secondary palate in 84% of the treated mice that were born. Moreover, the antisense ODN treatment resulted in a failure of apoptosis in the MEE between palatal shelves in physical contact in organ culture. We conclude that βig‐h3 expression in the MEE is stimulated by TGF‐β3, causes cell death, and consequently results in complete fusion of the apposed palatal shelves. J. Cell. Biochem. 107: 818–825, 2009. © 2009 Wiley‐Liss, Inc.     

GSKIP,an inhibitor of GSK3β, mediates the N‐cadherin/β‐catenin pool in the differentiation of SH‐SY5Y cells

Emerging evidence has shown that GSK3β plays a pivotal role in regulating the specification of axons and dendrites. Our previous study has shown a novel GSK3β interaction protein (GSKIP) able to negatively regulate GSK3β in Wnt signaling pathway. To further characterize how GSKIP functions in neurons, human neuroblastoma SH‐SY5Y cells treated with retinoic acid (RA) to differentiate to neuron‐like cells was used as a model. Overexpression of GSKIP prevents neurite outgrowth in SH‐SY5Y cells. GSKIP may affect GSK3β activity on neurite outgrowth by inhibiting the specific phosphorylation of tau (ser396). GSKIP also increases β‐catenin in the nucleus and raises the level of cyclin D1 to promote cell‐cycle progression in SH‐SY5Y cells. Additionally, overexpression of GSKIP downregulates N‐cadherin expression, resulting in decreased recruitment of β‐catenin. Moreover, depletion of β‐catenin by small interfering RNA, neurite outgrowth is blocked in SH‐SY5Y cells. Altogether, we propose a model to show that GSKIP regulates the functional interplay of the GSK3β/β‐catenin, β‐catenin/cyclin D1, and β‐catenin/N‐cadherin pool during RA signaling in SH‐SY5Y cells. J. Cell. Biochem. 108: 1325–1336, 2009. © 2009 Wiley‐Liss, Inc.     

Activation of Smad‐mediated TGF‐β signaling triggers epithelial–mesenchymal transitions in murine cloned corneal progenitor cells

Epithelial–mesenchymal transition (EMT), via activation of Wnt signaling, is prevailing in embryogenesis, but postnatally it only occurs in pathological processes, such as in tissue fibrosis and tumor metastasis. Our prior studies led us to speculate that EMT might be involved in the loss of limbal epithelial stem cells in explant cultures. To examine this hypothesis, we successfully grew murine corneal/limbal epithelial progenitors by prolonging the culture time and by seeding at a low density in a serum‐free medium. Single cell‐derived clonal growth was accompanied by a gradient of Wnt signaling activity, from the center to the periphery, marked by a centrifugal loss of E‐cadherin and β‐catenin from intercellular junctions, coupled with nuclear translocation of β‐catenin and LEF‐1. Large‐colony‐forming efficiency at central location of colony was higher than peripheral location. Importantly, there was also progressive centrifugal differentiation, with positive K14 keratin expression and the loss of p63 and PCNA nuclear staining, and irreversible EMT, evidenced by cytoplasmic expression of α‐SMA and nuclear localization of S100A4; and by nuclear translocation of Smad4. Furthermore, cytoplasmic expression of α‐SMA was promoted by high‐density cultures and their conditioned media, which contained cell density‐dependent levels of TGF‐β1, TGF‐β2, GM‐CSF, and IL‐1α. Exogenous TGF‐β1 induced α‐SMA positive cells in a low‐density culture, while TGF‐β1 neutralizing antibody partially inhibited α‐SMA expression in a high‐density culture. Collectively, these results indicate that irreversible EMT emerges in the periphery of clonal expansion where differentiation and senescence of murine corneal/limbal epithelial progenitors occurs as a result of Smad‐mediated TGF‐β‐signaling. J. Cell. Physiol. 228: 225–234, 2013. © 2012 Wiley Periodicals, Inc.     

Transforming Growth Factor‐β1 Modulates the Expression of Syndecan‐4 in Cultured Vascular Endothelial Cells in a Biphasic Manner

Proteoglycans are macromolecules that consist of a core protein and one or more glycosaminoglycan side chains. Previously, we reported that transforming growth factor‐β₁ (TGF‐β₁) regulates the synthesis of a large heparan sulfate proteoglycan, perlecan, and a small leucine‐rich dermatan sulfate proteoglycan, biglycan, in vascular endothelial cells depending on cell density. Recently, we found that TGF‐β₁ first upregulates and then downregulates the expression of syndecan‐4, a transmembrane heparan sulfate proteoglycan, via the TGF‐β receptor ALK5 in the cells. In order to identify the intracellular signal transduction pathway that mediates this modulation, bovine aortic endothelial cells were cultured and treated with TGF‐β₁. Involvement of the downstream signaling pathways of ALK5—the Smad and MAPK pathways—in syndecan‐4 expression was examined using specific siRNAs and inhibitors. The data indicate that the Smad3–p38 MAPK pathway mediates the early upregulation of syndecan‐4 by TGF‐β₁, whereas the late downregulation is mediated by the Smad2/3 pathway. Multiple modulations of proteoglycan synthesis may be involved in the regulation of vascular endothelial cell functions by TGF‐β₁. J. Cell. Biochem. 118: 2009–2017,2017. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.     

Blockade of JAK2 activity suppressed accumulation of β‐catenin in leukemic cells

The Wnt/β‐catenin pathway has been implicated in leukemogenesis. We found β‐catenin abnormally accumulated in both human acute T cell leukemia Jurkat cells and human erythroleukemia HEL cells. β‐Catenin can be significantly down‐regulated by the Janus kinase 2 specific inhibitor AG490 in these two cells. AG490 also reduces the luciferase activity of a reporter plasmid driven by LEF/β‐catenin promoter. Similar results were observed in HEL cells infected with lentivirus containing shRNA against JAK2 gene. After treatment with 50 µM AG490 or shRNA, the mRNA expression levels of β‐catenin, APC, Axin, β‐Trcp, GSK3α, and GSK3β were up‐regulated within 12–16 h. However, only the protein levels of GSK3β and β‐Trcp were found to have increased relative to untreated cells. Knockdown experiments revealed that the AG490‐induced inhibition of β‐catenin can be attenuated by shRNA targeting β‐TrCP. Taken together; these results suggest that β‐Trcp plays a key role in the cross‐talk between JAK/STAT and Wnt/β‐catenin signaling in leukemia cells. J. Cell. Biochem. 111: 402–411, 2010. © 2010 Wiley‐Liss, Inc.     

Transforming growth factor‐beta 1 (TGF‐β1) induces angiogenesis through vascular endothelial growth factor (VEGF)‐mediated apoptosis

VEGF and TGF‐β1 induce angiogenesis but have opposing effects on endothelial cells. VEGF protects endothelial cells from apoptosis; TGF‐β1 induces apoptosis. We have previously shown that VEGF/VEGF receptor‐2 (VEGFR2) signaling mediates TGF‐β1 induction of apoptosis. This finding raised an important question: Does this mechanism stimulate or inhibit angiogenesis? Here we report that VEGF‐mediated apoptosis is required for TGF‐β1 induction of angiogenesis. In vitro the apoptotic effect of TGF‐β1 on endothelial cells is rapid and followed by a long period in which the cells are refractory to apoptosis induction by TGF‐β1. Inhibition of VEGF/VEGFR2 signaling abrogates formation of cord‐like structures by TGF‐β1 with an effect comparable to that of z‐VAD, an apoptosis inhibitor. Similarly, genetic deficiency of VEGF abolishes TGF‐β1 upregulation of endothelial cell differentiation and formation of vascular structures in embryoid bodies. In vivo TGF‐β1 induces endothelial cell apoptosis as rapidly as in vitro. Inhibition of VEGF blocks TGF‐β1 induction of both apoptosis and angiogenesis, an effect similar to that of z‐VAD. Thus, TGF‐β1 induction of angiogenesis requires a rapid and transient apoptotic effect mediated by VEGF/VEGFR2. This novel, unexpected role of VEGF and VEGFR2 indicates VEGF‐mediated apoptosis as a potential target to control angiogenesis. J. Cell. Physiol. 219: 449–458, 2009. © 2009 Wiley‐Liss, Inc.     

TGF‐β Stimulates Expression of Chondroitin Polymerizing Factor in Nucleus Pulposus Cells Through the Smad3, RhoA/ROCK1, and MAPK Signaling Pathways

The enzyme chondroitin polymerizing factor (ChPF) is primarily involved in extension of the chondroitin sulfate backbone required for the synthesis of sulfated glycosaminoglycan (sGAG). Transforming growth factor beta (TGF‐β) upregulates sGAG synthesis in nucleus pulposus cells; however, the mechanisms mediating this induction are incompletely understood. Our study demonstrated that ChPF expression was negatively correlated with the grade of degenerative intervertebral disc disease. Treatment of nucleus pulposus cells with TGF‐β induced ChPF expression and enhanced Smad2/3, RhoA/ROCK activation, and the JNK, p38, and ERK1/2 MAPK signaling pathways. Selective inhibitors of Smad2/3, RhoA or ROCK1/2, and knockdown of Smad3 and ROCK1 attenuated ChPF expression and sGAG synthesis induced by TGF‐β. In addition, we showed that RhoA/ROCK1 signaling upregulated ChPF via activation of the JNK pathway but not the p38 and ERK1/2 signaling pathways. Moreover, inhibitors of JNK, p38 and ERK1/2 activity also blocked ChPF expression and sGAG synthesis induced by TGF‐β in a Smad3‐independent manner. Collectively, our data suggest that TGF‐β stimulated the expression of ChPF and sGAG synthesis in nucleus pulposus cells through Smad3, RhoA/ROCK1 and the three MAPK signaling pathways. J. Cell. Biochem. 119: 566–579, 2018. © 2017 Wiley Periodicals, Inc.     

Biglycan Intensifies ALK5–Smad2/3 Signaling by TGF‐β1 and Downregulates Syndecan‐4 in Cultured Vascular Endothelial Cells

Proteoglycans are macromolecules that consist of a core protein and one or more glycosaminoglycan side chains. A small leucine‐rich dermatan sulfate proteoglycan, biglycan, is one of the predominant types of proteoglycans synthesized by vascular endothelial cells; however, the physiological functions of biglycan are not completely understood. In the present study, bovine aortic endothelial cells in culture were transfected with small interfering RNAs for biglycan, and the expression of other proteoglycans was examined. Transforming growth factor‐β₁ signaling was also investigated, because the interaction of biglycan with cytokines has been reported. Biglycan was found to form a complex with either transforming growth factor‐β₁ or the transforming growth factor‐β₁ type I receptor, ALK5, and to intensify the phosphorylation of Smad2/3, resulting in a lower expression of the transmembrane heparan sulfate proteoglycan, syndecan‐4. This is the first report to clarify the function of biglycan as a regulatory molecule of the ALK5–Smad2/3 TGF‐β₁ signaling pathway that mediates the suppression of syndecan‐4 expression in vascular endothelial cells. J. Cell. Biochem. 118: 1087–1096, 2017. © 2016 Wiley Periodicals, Inc.     

Calcitonin gene‐related peptide (CGRP) inhibits apoptosis in human osteoblasts by β‐catenin stabilization

Transgenic mice over‐expressing calcitonin gene‐related peptide (CGRP) in osteoblasts have increased bone density due to increased bone formation, thus suggesting that CGRP plays a role in bone metabolism. In this study we determined the relationship between CGRP, the canonical Wnt signaling and apoptosis in human osteoblasts (hOBs) in consideration of the well‐documented involvement of this pathway in bone cells. Primary cultures of hOBs were treated with CGRP 10^?8 M. Levels of β‐catenin, which is the cytoplasmic protein mediator of canonical Wnt signaling, and mRNA were determined. CGRP increases both the expression and the levels of cytoplasmic β‐catenin by binding to its receptor, as this effect is blocked by the antagonist CGRP_8–37. This facilitatory action on β‐catenin appears to be mediated by the inhibition of the enzyme GSK‐3β via protein kinase A (PKA) activation. GSK‐3β is a glycogen synthase kinase that, by phosphorylating β‐catenin, promotes its degradation by the proteosomal machinery. Moreover, the peptide is able to inhibit hOBs apoptosis stimulated by dexamethasone or by serum deprivation, possibly through the accumulation of β‐catenin, since the inhibitor of PKA activity H89 partially prevents the antiapoptotic effect of the peptide. In conclusion CGRP, released by nerve fibers, exerts its anabolic action on bone cells by stimulating canonical Wnt signaling and by inhibiting hOBs apoptosis, thus favoring local bone regeneration. J. Cell. Physiol. 225: 701–708, 2010. © 2010 Wiley‐Liss, Inc.     

β‐Catenin—A supporting role in the skeleton

In the last 5 years a role for β‐catenin in the skeleton has been cemented. Beginning with mutations in the Lrp5 receptor that control β‐catenin canonical downstream signals, and progressing to transgenic models with bone‐specific alteration of β‐catenin, research has shown that β‐catenin is required for normal bone development. A cell critical to bone in which β‐catenin activity determines function is the marrow‐derived mesenchymal stem cell (MSC), where sustained β‐catenin prevents its distribution into adipogenic lineage. β‐Catenin actions are less well understood in mature osteoblasts: while β‐catenin contributes to control of osteoclastic bone resorption via alteration of the osteoprotegerin/RANKL ratio, a specific regulatory role during osteoblast bone synthesis has not yet been determined. The proven ability of mechanical factors to prevent β‐catenin degradation and induce nuclear translocation through Lrp‐independent mechanisms suggests processes by which exercise might modulate bone mass via control of lineage allocation, in particular, by preventing precursor distribution into the adipocyte pool. Effects resulting from mechanical activation of β‐catenin in mature osteoblasts and osteocytes likely modulate bone resorption, but whether β‐catenin is involved in osteoblast synthetic function remains to be proven for both mechanical and soluble mediators. As β‐catenin appears to support the downstream effects of multiple osteogenic factors, studies clarifying when and where β‐catenin effects occur will be relevant for translational approaches aimed at preventing bone loss and terminal adipogenic conversion. J. Cell. Biochem. 110: 545–553, 2010. © 2010 Wiley‐Liss, Inc.     

Phosphorylation of PPP(S/T)P motif of the free LRP6 intracellular domain is not required to activate the Wnt/β‐catenin pathway and attenuate GSK3β activity

The canonical Wnt/β‐catenin signaling pathway plays a critical role in numerous physiological and pathological processes. LRP6 is an essential co‐receptor for Wnt/β‐catenin signaling; as transduction of the Wnt signal is strongly dependent upon GSK3β‐mediated phosphorylation of multiple PPP(S/T)P motifs within the membrane‐anchored LRP6 intracellular domain. Previously, we showed that the free LRP6 intracellular domain (LRP6‐ICD) can activate the Wnt/β‐catenin pathway in a β‐catenin and TCF/LEF‐1 dependent manner, as well as interact with and attenuate GSK3β activity. However, it is unknown if the ability of LRP6‐ICD to attenuate GSK3β activity and modulate activation of the Wnt/β‐catenin pathway requires phosphorylation of the LRP6‐ICD PPP(S/T)P motifs, in a manner similar to the membrane‐anchored LRP6 intracellular domain. Here we provide evidence that the LRP6‐ICD does not have to be phosphorylated at its PPP(S/T)P motif by GSK3β to stabilize endogenous cytosolic β‐catenin resulting in activation of TCF/LEF‐1 and the Wnt/β‐catenin pathway. LRP6‐ICD and a mutant in which all 5 PPP(S/T)P motifs were changed to PPP(A)P motifs equivalently interacted with and attenuated GSK3β activity in vitro, and both constructs inhibited the in situ GSK3β‐mediated phosphorylation of β‐catenin and tau to the same extent. These data indicate that the LRP6‐ICD attenuates GSK3β activity similar to other GSK3β binding proteins, and is not a result of it being a GSK3β substrate. Our findings suggest the functional and regulatory mechanisms governing the free LRP6‐ICD may be distinct from membrane‐anchored LRP6, and that release of the LRP6‐ICD may provide a complimentary signaling cascade capable of modulating Wnt‐dependent gene expression. J. Cell. Biochem. 108: 886–895, 2009. © 2009 Wiley‐Liss, Inc.