Stretch-induced phosphorylation of ERK1/2 depends on differentiation stage of osteoblasts

The goal of this study was to investigate the effect of mechanical loading on osteoblasts and extracellular signal-regulated kinase (ERK1/2) signaling in relation to osteoblast differentiation and mineralization. A human osteoblast cell line (SV-HFO) was triggered to differentiate to the advanced state of mineralization by addition of the osteogenic factors dexamethasone and beta-glycerophosphate. Osteoblasts were subjected to cyclic, equibiaxial stretch for 5, 15, or 60 min at different stages of differentiation (days 7, 14, and 21). Baseline (static) phosphorylated ERK1/2 and total ERK1/2 levels gradually increased during osteoblast differentiation. Cyclic stretch induced a rapid increase in ERK1/2 phosphorylation with a maximum between 5 and 15 min. Prolonged stretching for 60 min resulted in a decrease of phosphorylated ERK1/2 towards baseline level, suggesting a desensitization mechanism. The effect of stretch on ERK1/2 phosphorylation was strongest at later stages of differentiation (days 14 and 21). At day 21, the increase of ERK1/2 phosphorylation in response to stretch was significantly lower in non-differentiating than in differentiating osteoblasts. This could not be explained by differences in cell density, but did correlate with the formation of extracellular matrix, collagen fibrils. Mineralization of the extracellular matrix did not lead to a further increase of ERK1/2 phosphorylation. In conclusion, the current study demonstrates that the extent of activation of the ERK1/2 pathway is dependent on the differentiation or functional stage of the osteoblast. The presence of an extracellular matrix, but not per se mineralization, seems to be the predominant determinant of osteoblastic response to strain.     

IGF1 regulates RUNX1 expression via IRS1/2: Implications for antler chondrocyte differentiation

Although IGF1 is important for the proliferation and differentiation of chondrocytes, its underlying molecular mechanism is still unknown. Here we addressed the physiologic function of IGF1 in antler cartilage and explored the interplay of IGF1, IRS1/2 and RUNX1 in chondrocyte differentiation. The results showed that IGF1 was highly expressed in antler chondrocytes. Exogenous rIGF1 could increase the proliferation of chondrocytes and cell proportion in the S phase, whereas IGF1R inhibitor PQ401 abrogated the induction by rIGF1. Simultaneously, IGF1 could stimulate the expression of IHH which was a well-known marker for prehypertrophic chondrocytes. Further analysis evidenced that IGF1 regulated the expression of IRS1/2 whose silencing resulted in a rise of IHH mRNA levels, but the regulation was impeded by PQ401. Knockdown of IRS1 or IRS2 with specific siRNA could greatly enhance rIGF1-induced chondrocyte differentiation and reduce the expression of RUNX1. Extraneous rRUNX1 might rescue the effects of IRS1 or IRS2 siRNA on the differentiation. In antler chondrocytes, IGF1 played a role in modulating the expression of RUNX1 through IGF1R. Moreover, attenuation of RUNX1 expression advanced the differentiation elicited by rIGF1, while administration of rRUNX1 to chondrocytes treated with IGF1 siRNA or PQ401 reduced their differentiation. Additionally, siRNA-mediated downregulation of IRS1 or IRS2 in the chondrocytes impaired the interaction between IGF1 and RUNX1. Collectively, IGF1 could promote the proliferation and differentiation of antler chondrocytes. Furthermore, IRS1/2 might act downstream of IGF1 to regulate chondrocyte differentiation through targeting RUNX1.     

Polycystin-1 downregulation induces ERK-dependent mTOR pathway activation in a cellular model of psoriasis

Psoriatic plaques tend to localize to the knees and elbows, areas that are particularly subject to mechanical stress resulting from bending and friction. Moreover, plaques often develop at sites of mechanical trauma or injury (Koebner phenomenon). Nevertheless, mechanotransduction has never been linked to psoriasis. Polycystins (polycystin-1, PC1; polycystin-2, PC2) are mechanosensitive molecules that function as key regulators of cellular mechanosensitivity and mechanotransduction. The aim of this in vitro study was to investigate the role of polycystins in the development of psoriasis. We showed that PC1 knockdown in HaCaT cells led to an elevated mRNA expression of psoriasis-related biomarkers Ki-67, IL-6, TNF-α, VEGF and Bcl-2, while PC1 functional inhibition was accompanied by increased cell proliferation and migration of HaCaT cells. In addition, PC1 knockdown via siRNA in HaCaT cells was followed by activation of critical molecules of the mTOR and MAPK pathways and this mTOR pathway activation was ERK-dependent. Furthermore, loss of PC1 protein expression and elevated levels of activated mTOR substrates were also observed in human samples of psoriatic plaques. Overall, our study suggests that the PC1/ERK/mTOR signaling axis represents a novel potential mechanism in psoriasis pathogenesis.     

Glutamate accelerates RPE cell proliferation through ERK1/2 activation via distinct receptor-specific mechanisms

The proliferation and migration of Retinal Pigment Epithelium cells resulting from an epithelial-mesenchymal transition plays a key role in proliferative vitreoretinopathy, which leads to retinal detachment and the loss of vision. In neurons, glutamate has been shown to activate the Ras/Raf/MEK/ERK cascade, which participates in the regulation of proliferation, differentiation, and survival processes. Although glutamate-stimulation and the activation of ERK1/2 by different stimuli have been shown to promote RPE cell proliferation, the signaling pathway(s) linking these effects has not been established. We analyzed the molecular mechanisms leading to glutamate-induced proliferation by determining ERK1/2 and CREB phoshporylation in chick RPE cells in primary culture and the human-derived RPE cell line ARPE-19. This study shows for the first time, that glutamate promotes RPE cell proliferation by activating two distinct signaling pathways linked to selective glutamate receptor subtypes. Results demonstrate that glutamate stimulates RPE cell proliferation as well as ERK and CREB phosphorylation. These effects were mimicked by the mGluR agonist ACPD and by NMDA, and were prevented by the respective receptor inhibitors MCPG and MK-801, indicating a cause-effect relationship between these processes. Whereas mGluR promoted proliferation by activating the MEK/ERK/CREB cascade, NMDA stimulated proliferation through the MEK-independent activation of Ca(2+)/calmodulin-dependent kinases. The blockage of both signaling pathways to proliferation by KN-62 suggests the involvement of CaMKs in the control of glutamate-induced proliferation at a common step, downstream of CREB, possibly the regulation of cell cycle progression. Based on these findings, the participation of glutamate in the development of PVR can be considered.     

Cholesterol depletion induces ANTXR2-dependent activation of MMP-2 via ERK1/2 phosphorylation in neuroglioma U251 cell

Cholesterol is a critical component of lipid rafts implicated in regulating multiple signal transduction. The anthrax toxin receptor 2 (ANTXR2) is a type I membrane protein acting as the second receptor for the anthrax toxin. In this study, we first investigated the association between cholesterol and ANTXR2. We provided the evidence that cholesterol depletion by methyl-beta-cyclodextrin (MβCD) promoted ANTXR2 expression in U251 neuroglioma cell, which was reversed by cholesterol supplement. MβCD-induced ANTXR2 up-regulation contributed to ERK1/2 phosphorylation, which was responsible for MT1-MMP and MMP-2 activation. Our data suggested that cellular cholesterol regulated ANTXR2-dependent activation of MMP-2 via ERK1/2 phosphorylation in neuroglioma U251 cell.     

Effects of insulin and insulin‐like growth factor 1 on osteoblast proliferation and differentiation: differential signalling via Akt and ERK

Insulin and insulin‐like growth factor 1 (IGF‐1) are evolutionarily conserved hormonal signalling molecules, which influence a wide array of physiological functions including metabolism, growth and development. Using genetic mouse studies, both insulin and IGF‐1 have been shown to be anabolic agents in osteoblasts and bone development primarily through the activation of Akt and ERK signalling pathways. In this study, we examined the temporal signalling actions of insulin and IGF‐1 on primary calvarial osteoblast growth and differentiation. First, we observed that the IGF‐1 receptor expression decreases whereas insulin receptor expression increases during osteoblast differentiation. Subsequently, we show that although both insulin and IGF‐1 promote osteoblast differentiation and mineralization in vitro, IGF‐1, but not insulin, can induce osteoblast proliferation. The IGF‐1‐induced osteoblast proliferation was mediated via both MAPK and Akt pathways because the IGF‐1‐mediated cell proliferation was blocked by U0126, an MEK/MAPK inhibitor, or LY294002, a PI3‐kinase inhibitor. Osteocalcin, an osteoblast‐specific protein whose expression corresponds with osteoblast differentiation, was increased in a dose‐ and time‐dependent manner after insulin treatment, whereas it was decreased with IGF‐1 treatment. Moreover, insulin treatment dramatically induced osteocalcin promoter activity, whereas IGF‐1 treatment significantly inhibited it, indicating direct effect of insulin on osteocalcin synthesis. Copyright © 2012 John Wiley & Sons, Ltd.     

Polycystin‐1 regulates cell proliferation and migration through AKT/mTORC2 pathway in a human craniosynostosis cell model

Craniosynostosis is the premature fusion of skull sutures and has a severe pathological impact on childrens’ life. Mechanical forces are capable of triggering biological responses in bone cells and regulate osteoblastogenesis in cranial sutures, leading to premature closure. The mechanosensitive proteins polycystin‐1 (PC1) and polycystin‐2 (PC2) have been documented to play an important role in craniofacial proliferation and development. Herein, we investigated the contribution of PC1 to the pathogenesis of non‐syndromic craniosynostosis and the associated molecular mechanisms. Protein expression of PC1 and PC2 was detected in bone fragments derived from craniosynostosis patients via immunohistochemistry. To explore the modulatory role of PC1 in primary cranial suture cells, we further abrogated the function of PC1 extracellular mechanosensing domain using a specific anti‐PC1 IgPKD1 antibody. Effect of IgPKD1 treatment was evaluated with cell proliferation and migration assays. Activation of PI3K/AKT/mTOR pathway components was further detected via Western blot in primary cranial suture cells following IgPKD1 treatment. PC1 and PC2 are expressed in human tissues of craniosynostosis. PC1 functional inhibition resulted in elevated proliferation and migration of primary cranial suture cells. PC1 inhibition also induced activation of AKT, exhibiting elevated phospho (p)‐AKT (Ser473) levels, but not 4EBP1 or p70S6K activation. Our findings indicate that PC1 may act as a mechanosensing molecule in cranial sutures by modulating osteoblastic cell proliferation and migration through the PC1/AKT/mTORC2 cascade with a potential impact on the development of non‐syndromic craniosynostosis.     

Sema4D/PlexinB1 promotes endothelial differentiation of dental pulp stem cells via activation of AKT and ERK1/2 signaling

Inducing of dental pulp stem cells (DPSCs) into endothelial cells (ECs) to prevascularize pulp tissue constructs may offer a novel and viable approach for enhancing pulp regeneration. However, there are numerous challenges in current methods for the acquisition of sufficient translational ECs. It was known that Sema4D/PlexinB1 signaling exerts profound effects on enhancing vascular endothelial growth factor (VEGF) secretion and angiogenesis. Whether Sema4D/PlexinB1 could regulate endothelial differentiation of DPSCs is not yet investigated. In this study, when DPSCs were treated with Sema4D (2 μg/mL), ECs-specific (VEGFR1, VEGFR2, CD31, and vWF), and angiogenic genes and proteins were significantly upregulated. The induced ECs exhibited similar endothelial vessel formation ability to that of human umbilical vein endothelial cells (HUVECs). Furthermore, phosphorylation of AKT increased dramatically within 5 minutes (from 0.93 to 21.8), while p-ERK1/2 was moderately elevated (from 0.94 to 2.65). In summary, our results demonstrated that Sema4D/PlexinB1 signaling induces endothelial differentiation of DPSCs. The interactions of Sema4D, VEGF, ANGPTL4, ANG1, and HIF-1α may play a crucial role in mediating the differentiation process.     

Resveratrol enhances proliferation and osteoblastic differentiation in human mesenchymal stem cells via ER-dependent ERK1/2 activation

In the present study, we investigated the in vitro effect of resveratrol (RSVL), a polyphenolic phytoestrogen, on cell proliferation and osteoblastic maturation in human bone marrow-derived mesenchymal stem cell (HBMSC) cultures. RSVL (10⁻⁸–10⁻⁵ M) increased cell growth dose-dependently, as measured by [³H]-thymidine incorporation, and stimulated osteoblastic maturation as assessed by alkaline phosphatase (ALP) activity, calcium deposition into the extracellular matrix, and the expression of osteoblastic markers such as RUNX2/CBFA1, Osterix and Osteocalcin in HBMSCs cell cultures. Further studies found that RSVL (10⁻⁶ M) resulted in a rapid activation of both extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) signaling in HBMSCs cultures. The effects of RSVL were mimicked by 17β-estrodial (10⁻⁸ M) and were abolished by estrogen receptor (ER) antagonist ICI182780. An ERK1/2 pathway inhibitor, PD98059, significantly attenuated RSVL-induced ERK1/2 phosphorylation, consistent with the reduction of cell proliferation and osteoblastic differentiation as well as expression of osteoblastic markers. In contrast, SB203580, a p38 MAPK pathway blocker, blocked RSVL-induced p38 phosphorylation, but resulted in an increase of cell proliferation and a more osteoblastic maturation. These data suggest that RSVL stimulates HBMSCs proliferation and osteoblastic differentiation through an ER-dependent mechanism and coupling to ERK1/2 activation.     

Overexpression of BMP-2 modulates morphology, growth, and gene expression in osteoblastic cells

Bone morphogenetic proteins (BMP) play a pivotal role in growth and differentiation of osteoblastic lineage cells. BMPs are potent stimulators of bone formation in various animal models. To understand the mechanism of BMP action in bone cells, we have investigated the effects of overexpression of the BMP-2 gene on proliferation and differentiation of UMR-106 rat osteosarcoma cells. A stable UMR-106 cell line overexpressing the BMP-2 gene was established by transfection of cells using a mammalian expression vector harboring human BMP-2 cDNA followed by G418 selection. After introduction of the BMP-2 gene, UMR-106 cells appeared more spindle-shaped in morphology compared to the predominantly cuboidal appearance of the parental cells. Overexpression of BMP-2 markedly inhibited proliferation as measured by cell counting and [3H]thymidine incorporation assays. Extracellular matrix (ECM) derived from cells overexpressing BMP-2 exhibited a less supportive effect on proliferation of UMR cells than did ECM derived from parental cells. Furthermore, cell-cell communication through gap junctions was reduced more than 50% as determined by nondisruptive fluorescent dye transfer assays. Overexpression of BMP-2 significantly stimulated expression of osteocalcin and alkaline phosphatase genes, indicating its role in osteoblastic differentiation. There was little effect on osteopontin gene expression.     

Distinct functions for ERK1 and ERK2 in cell migration processes during zebrafish gastrulation

The MAPKs are key regulatory signaling molecules in many cellular processes. Here we define differential functions for ERK1 and ERK2 MAPKs in zebrafish embryogenesis. Morpholino knockdown of ERK1 and ERK2 resulted in cell migration defects during gastrulation, which could be rescued by co-injection of the corresponding mRNA. Strikingly, Erk2 mRNA cross-rescued ERK1 knockdown, but erk1 mRNA was unable to compensate for ERK2 knockdown. Cell-tracing experiments revealed a convergence defect for ERK1 morphants without a severe posterior-extension defect, whereas ERK2 morphants showed a more severe reduction in anterior-posterior extension. These defects were primary changes in gastrulation cell movements and not caused by altered cell fate specification. Saturating knockdown conditions showed that the absence of FGF-mediated dual-phosphorylated ERK2 from the blastula margin blocked initiation of epiboly, actin and tubulin cytoskeleton reorganization processes and further arrested embryogenesis, whereas ERK1 knockdown had only a mild effect on epiboly progression. Together, our data define distinct roles for ERK1 and ERK2 in developmental cell migration processes during zebrafish embryogenesis.     

RUNX1 and RUNX2 transcription factors function in opposing roles to regulate breast cancer stem cells

Breast cancer stem cells (BCSCs) are competent to initiate tumor formation and growth and refractory to conventional therapies. Consequently BCSCs are implicated in tumor recurrence. Many signaling cascades associated with BCSCs are critical for epithelial-to-mesenchymal transition (EMT). We developed a model system to mechanistically examine BCSCs in basal-like breast cancer using MCF10AT1 FACS sorted for CD24 (negative/low in BCSCs) and CD44 (positive/high in BCSCs). Ingenuity Pathway Analysis comparing RNA-seq on the CD24^−/low versus CD24^+/high MCF10AT1 indicates that the top activated upstream regulators include TWIST1, TGFβ1, OCT4, and other factors known to be increased in BCSCs and during EMT. The top inhibited upstream regulators include ESR1, TP63, and FAS. Consistent with our results, many genes previously demonstrated to be regulated by RUNX factors are altered in BCSCs. The RUNX2 interaction network is the top significant pathway altered between CD24^−/low and CD24^+/high MCF10AT1. RUNX1 is higher in expression at the RNA level than RUNX2. RUNX3 is not expressed. While, human-specific quantitative polymerase chain reaction primers demonstrate that RUNX1 and CDH1 decrease in human MCF10CA1a cells that have grown tumors within the murine mammary fat pad microenvironment, RUNX2 and VIM increase. Treatment with an inhibitor of RUNX binding to CBFβ for 5 days followed by a 7-day recovery period results in EMT suggesting that loss of RUNX1, rather than increase in RUNX2, is a driver of EMT in early stage breast cancer. Increased understanding of RUNX regulation on BCSCs and EMT will provide novel insight into therapeutic strategies to prevent recurrence.     

Human lactoferrin exerts bi-directional actions on PC12 cell survival via ERK1/2 pathway

Human lactoferrin (hLF) is a member of the transferrin family and is found in most body fluids of human. Recent study showed that hLF played some roles in the regulation of cell growth. However, the biological function of hLF in the central nervous system and neuronal cells is still unclear. The MTT was used to assay cell viability, ELISA tests were used to assay caspase activities, and TUNEL staining was used to test the cytotoxicity of hLF to the cells. Our result showed that 700 microg/ml hLF significantly reduced the cell viability and increased the caspase 3 and 8 activities in PC12 neuronal cells. TUNEL staining further showed that 700 microg/ml hLF was cytotoxic to the PC12 through apoptosis-mediated pathway. In addition, 700 microg/ml hLF significantly decreased the protein expressions of phosphorylated extracellular-signal-regulated kinase 1/2 (ERK1/2) and Bcl-2 in PC12 cells, whereas 50 microg/ml hLF significantly increased the phosphorylation of ERK1/2 which could be specifically inhibited by PD98059. Furthermore, 50 microg/ml hLF could not only up-regulate the Bcl-2 expression but also protect PC12 cells from FasL-induced apoptosis. In conclusion, hLF plays a crucial role in the regulation of apoptosis and anti-apoptosis in PC12 neuronal cells via ERK1/2 phosphorylation pathway.     

Vitamin C induces periodontal ligament progenitor cell differentiation via activation of ERK pathway mediated by PELP1

The differentiation of periodontal ligament (PDL) progenitor cells is important for maintaining the homeostasis of PDL tissue and alveolar bone. Vitamin C (VC), a water-soluble nutrient that cannot be biosynthesized by humans, is vital for mesenchymal stem cells differentiation and plays an important role in bone remodeling. Therefore, the objective of this study was to determine the function and mechanism of VC in PDL progenitor cells osteogenic differentiation at the molecular level. We demonstrated that VC could induce the osteogenic differentiation and maturation of PDL progenitor cell without other osteogenic agents. During the process, VC preferentially activated ERK1/2 but did not affect JNK or p38. Co-treatment with ERK inhibitor effectively decreased the Vitamin C-induced expression of Runx2. ERK inhibitor also abrogated Vitamin C-induced the minimized nodules formation. PELP1, a nuclear receptor co-regulator, was up-regulated under VC treatment. PELP1 knockdown inhibited ERK phosphorylation. The overexpression of PELP1 had a positive relationship with Runx2 expression. Taken together, we could make a conclude that VC induces the osteogenic differentiation of PDL progenitor cells via PELP1-ERK axis. Our fi nding implies that VC may have a potential in the regeneration medicine and application to periodontitis treatment.     

Sodium arsenite-induced DAPK promoter hypermethylation and autophagy via ERK1/2 phosphorylation in human uroepithelial cells

Arsenic compounds or arsenicals are well-known toxic and carcinogenic agents. The toxic effects of arsenic that are of most concern to humans are those that occur from chronic, low-level exposure, and are associated with various human malignancies, including skin, lung and bladder cancers. In addition, arsenic could induce cell death, including apoptosis or autophagy in malignant cells. Previously, we have demonstrated that arsenite can induce autophagy and death-associated protein kinase (DAPK) promoter hypermethylation in the SV-40 immortalized human uroepithelial cell line (SV-HUC-1). However, the underlying mechanism of arsenite-induced autophagy is still unclear. In the present study, we demonstrate that arsenite can activate the extracellular signaling-regulated protein kinase 1/2 (ERK1/2) signaling pathway after treatment in SV-HUC-1 cells by using immunocytochemistry and Western blotting. In addition, our results also show an increase of autophagosomes was produced in arsenite-treated SV-HUC-1 cells by using electron microscopy. We found that, by incrementally increasing the dosages, microtubule-associated protein light chain 3B (LC3B) and Beclin-1 are important regulators for the formation of autophagosomes, in a dose-dependent manner. When the cells were pretreated with inhibitors 5-aza-CdR or U0126 for 24 h, the effect of arsenite on ERK1/2, LC3B, Beclin-1 and DAPK proteins expression is suppressed. Furthermore, our results support the notion that arsenite can induce the ERK1/2 signaling pathway to stimulate autophagy and DAPK promoter hypermethylation in human uroepithelial SV-HUC-1 cells. These findings may contribute to a better understanding of the carcinogenesis of arsenite.     

Myostatin regulates preadipocyte differentiation and lipid metabolism of adipocyte via ERK1/2

Myostatin is known as an inhibitor of muscle development, but its role in adipogenesis and lipid metabolism is still unclear, especially the underlying mechanisms. Here, we demonstrated that myostatin inhibited 3T3-L1 preadipocyte differentiation into adipocyte by suppressing C/EBPα (CCAAT/enhancer-binding protein α) and PPARγ (peroxisome-proliferator-activated receptor γ), also activated ERK1/2 (extracellular-signal-regulated kinase 1/2). Furthermore, myostatin enhanced the phosphorylation of HSL (hormone-sensitive lipase) and ACC (acetyl-CoA carboxylase) in fully differentiated adipocytes, as well as ERK1/2. Besides, we noted that myostatin markedly raised the levels of leptin and adiponectin release and mRNA expression during preadipocyte differentiation, but the levels were inhibited by myostatin treatments in fully differentiated adipocytes. These results suggested that myostatin suppressed 3T3-L1 preadipocyte differentiation and regulated lipid metabolism of mature adipocyte, in part, via activation of ERK1/2 signalling pathway.