Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.10.1 (ERK)
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The goal of this study was to determine the effects of hindlimb unloading (HU) on the ex vivo growth and the osteogenic potential of mesenchymal stem cells (MSCs) from the femurs of rats. Microgravity was simulated by 28-day HU in male Sprague-Dawley (SD) rats, and the bone marrow (BM) was collected from hindlimb femurs of HU or control (CTL) rats. MSCs were isolated from BM and cultured for eight passages. Then MSCs at passages 2, 4, and 8 were induced for osteogenesis or adipogenesis. The results revealed that HU decreased the osteogenic potential of MSCs and also decreased the expression of osteoblast gene marker mRNAs in cells induced by osteogenic conditions. Meanwhile, the expression of Runx2 mRNA and the phosphorylation of ERK were also decreased. There were no significant differences of osteoblast gene marker and Runx2 mRNA expression between cells induced from different passages of MSCs in UH rats. Under adipogenic conditions, HU increased both the adipogenic potential of MSCs and the expression of adipocytic gene marker mRNAs in induced cells. HU also increased the expression of PPAR gamma 2 mRNA, but with no effect on the phosphorylation of p38MAPK. The adipogenic potential of MSCs and the expression of adipocytic gene marker mRNAs in induced cells decreased along with cell cultures under normal gravity. This suggests that the normal gravity during in vitro MSC culture and the centrifugal force produced during cell harvest after each passage could decrease the adipogenic potential of MSCs, but could not reverse the effect of HU on the osteogenic potential of MSCs.
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PMID:Effects of hindlimb unloading on ex vivo growth and osteogenic/adipogenic potentials of bone marrow-derived mesenchymal stem cells in rats. 1871 Mar 46

The differentiation of osteoblasts from mesenchymal precursors requires a series of cell fate decisions controlled by a hierarchy of transcription factors. These include RUNX2, Osterix (OSX), ATF4 and a large number of nuclear coregulators. During bone development, initial RUNX2 expression coincides with the formation of mesenchymal condensations and precedes the branching of chondrogenic and osteogenic lineages. Given its central role in bone development, it is not surprising that RUNX2 is subject to a variety of controls. These include posttranslational modification, especially phosphorylation, and interactions with accessory nuclear factors. Specific examples of RUNX2 regulation to be reviewed include phosphorylation by the ERK/MAP kinase pathway and interactions with DLX5. RUNX2 is regulated via phosphorylation of critical serine residues in the proline/serine/threonine domain. In vivo, the transgenic expression of constitutively active MAP kinase in osteoblasts accelerated skeletal development, while a dominant-negative MAPK retarded development in a RUNX2-dependent manner. DLX5-RUNX2 complexes can be detected in osteoblasts and this interaction plays a critical role in maintaining osteoblast-specific expression of the bone sialoprotein gene. These studies allow us to begin understanding the complex mechanisms necessary to fine-tune bone formation as mesenchymal progenitors progress down the osteoblast lineage.
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PMID:Transcriptional regulation of osteoblasts. 1872 56

Metalloproteinase MT1-MMP is induced and Pro-MMP-2 up modulated early in rat preosteoblasts (ROB) set to differentiate. We here show that the induction of MMPs, accompanied by activation of Pro-MMP-2, occurs by 6 h of adhesion on endogenous extracellular matrix (ECM), Fibronectin (FN) and Collagen type I (CI). These events do not occur after adhesion on Collagen III (CIII), Vitronectin (VN) or BSA. Within the first hour on inducing substrata or plastic, FAK is unchanged and ERK(1,2), is activated, but this activation is not sufficient for MT1-MMP induction. The function of p38 MAPK and PTKs is not required for the induction by substrata of MMPs. Six hours after plating preosteoblasts on MMP-inducing substrata, complexes of beta1 integrin with MT1-MMP are formed, that contain integrin dimers specifically engaged by the substratum, alpha4 and alpha5 chains for cells plated on FN, and alpha2 chain for cells plated on CI and ECM. Induction of MT1-MMP and its expression during osteogenesis pleiotropically regulate alkaline phosphatase (AP) expression. During differentiation, variant clones derived from preosteoblasts and MMPs-over-expressing osteoblasts show high MT1-MMP level associated with high AP level both persisting in time, while inhibition of MMPs is accompanied by inhibition of AP. Up or down modulation of AP, transcriptionally or by inhibition of the enzyme activity, has no effect on level or timing of expression of MT1-MMP and Pro-MMP-2. The persistence in expression of MT1-MMP during differentiation, and the associated persistence in expression of AP, as well as their inhibition, both impair the formation of nodules and mineral deposition. A transient pattern of expression of MT1-MMP is required for the establishment of nodules, and MT1-MMP decrease is permissive for nodule mineralization. The expression of AP is required for nodule formation and its level modulates the mineralization. MT1-MMP has multiple functions and is implicated in multiple steps of the differentiation process, acting to regulate homeostasis of the osteogenic differentiation.
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PMID:Role of MT1-MMP in the osteogenic differentiation. 1902 88

Mesenchymal stem cells (MSCs) are able to differentiate into several lineages including osteoblasts. The signaling mechanisms involved in the osteogenic differentiation of MSCs are however not fully understood. We investigated the role of fibroblast growth factor receptor 2 (FGFR2) in osteoblast committment and differentiation of murine mesenchymal C3H10T1/2 cells stably transfected with wild type (WT) or activated FGFR2 due to Apert S252W genetic mutation (MT). WT FGFR2 slightly increased, whereas MT FGFR2 strongly increased, FGFR2 tyrosine phosphorylation, indicating activation of the receptor. WT and MT FGFR2 increased C3H10T1/2 cell proliferation but not survival. Both WT and MT FGFR2 increased early and late osteoblast gene expression and matrix mineralization. Forced expression of WT and MT FGFR2 also increased osteoblast gene expression in MC3T3-E1 calvaria osteoblasts. In both cell types, MT FGFR2 was more effective than WT FGFR2. In contrast, WT and MT FGFR2 decreased adipocyte differentiation of C3H10T1/2 cells. WT and MT FGFR2 induced ERK1/2 but not JNK or PI3K/AKT phosphorylation. MT, but not WT, also increased protein kinase C (PKC) activity. Pharmacological inhibition of ERK1/2 prevented cell proliferation induced by WT and MT FGFR2. Using dominant-negative ERK and PKCalpha vectors, we demonstrated that WT and MT FGFR2 promoted osteoblast gene expression through ERK1/2 and PKCalpha signaling, respectively. This study identifies FGFR2 as a novel regulatory molecule that promotes osteogenic differentiation in murine MSCs. The promoting effect of WT and MT FGFR2 is mediated by ERK1/2 and PKCalpha pathways that play essential and distinct roles in FGFR2-induced osteogenic differentiation of mesenchymal cells.
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PMID:Fibroblast growth factor receptor 2 promotes osteogenic differentiation in mesenchymal cells via ERK1/2 and protein kinase C signaling. 1911 54

The sol-to-gel transition occurring at around body temperature makes the MPEG-PCL diblock copolymer an ideal candidate material for use as an injectable in situ-forming gel containing human adipose tissue-derived stem cells (hADSCs). The sol can be prepared at room temperature, and the gel forms at body temperature. Solutions of the copolymer containing hADSCs and osteogenic factors injected into rats formed gel scaffolds at the injection sites. The gels thus formed showed the interconnective pore structure required to support growth, proliferation, and differentiation of hADSCs. Bromodeoxyuridine-labeled hADSCs were confirmed to be present in gels formed in vivo. Bone formation was observed only in gel implants containing both hADSCs and osteogenic factors. Subcutaneous implantation of the in situ-forming gel scaffold demonstrated that hADSCs embedded in the gel stimulated much lower host tissue responses than did the gel alone, probably because of the unique immunomodulatory properties of hADSCs. In conclusion, our data on hADSCs embedded in an in situ gel scaffold suggest that this formulation may provide numerous benefits as a noninvasive alternative for tissue-engineered bone formation.
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PMID:In vivo osteogenic differentiation of human adipose-derived stem cells in an injectable in situ-forming gel scaffold. 1913 93

CD34 is frequently used as a marker of adipose-derived stem/stromal/progenitor cells (ASCs). However, CD34 expression in human ASCs (hASCs) decreases over time in culture, and the implications of this remain unclear. In this study, we sorted shortly-cultured hASCs into CD34+ and CD34- fractions and compared their biological functions. Results indicated that CD34+ hASCs were more proliferative and had a greater ability to form colonies. In contrast, CD34- cells showed a greater ability for differentiation into adipogenic and osteogenic lineages. Both CD34+ and CD34- cells showed similar abilities in migration and capillary formation in response to growth factors. Expression levels of endothelial progenitor markers (Flk-1, FLT1, and Tie-2) were higher in CD34+ cells, whereas those of pericyte markers (CD146, NG2, and alpha-smooth muscle actin) were higher in CD34- cells. Both CD34+ and CD34- cells showed similar expression levels of vascular endothelial growth factor and hepatocyte growth factor, although hypoxia affected the expression levels. Together these results suggest that CD34 expression in hASCs may correlate with replicative capacity, differentiation potentials, expression profiles of angiogenesis-related genes, and immaturity or stemness of the cells. Loss of CD34 expression may be related to the physiological process of commitment and/or differentiation from immature status into specific lineages such as adipose, bone, or smooth muscle.
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PMID:Functional implications of CD34 expression in human adipose-derived stem/progenitor cells. 1922 22

Although ex vivo expanded mesenchymal stem cells (MSC) have been used in numerous studies, the molecular signature and in vivo distribution status of MSC remain unknown. To address this matter, we identified numerous human MSC-characteristic genes--including nine transcription factor genes--using DNA microarray and real-time RT-PCR analyses: Most of the MSC-characteristic genes were down-regulated 24 h after incubation with osteogenesis-, chondrogenesis- or adipogenesis-induction medium, or 48-72 h after knockdown of the nine transcription factors. Furthermore, knockdowns of ETV1, ETV5, FOXP1, GATA6, HMGA2, SIM2 or SOX11 suppressed the self-renewal capacity of MSC, whereas those of FOXP1, SOX11, ETV1, SIM2 or PRDM16 reduced the osteogenic- and/or adipogenic potential. In addition, immunohistochemistry using antibodies for the MSC characteristic molecules--including GATA6, TRPC4, FLG and TGM2--revealed that MSC-like cells were present near the endosteum and in the interior of bone marrow of adult mice. These findings indicate that MSC synthesize a set of MSC markers in vitro and in vivo, and that MSC-characteristic transcription factors are involved in MSC stemness regulation.
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PMID:Identification of mesenchymal stem cell (MSC)-transcription factors by microarray and knockdown analyses, and signature molecule-marked MSC in bone marrow by immunohistochemistry. 1922 1

Heparan sulfate proteoglycans cooperate with basic fibroblast growth factor (bFGF/FGF2) signaling to control osteoblast growth and differentiation, as well as metabolic functions of osteoblasts. FGF2 signaling modulates the expression and activity of Runt-related transcription factor 2 (Runx2/Cbfa1), a key regulator of osteoblast proliferation and maturation. Here, we have characterized novel Runx2 target genes in osteoprogenitors under conditions that promote growth arrest while not yet permitting sustained phenotypic maturation. Runx2 enhances expression of genes related to proteoglycan-mediated signaling, including FGF receptors (e.g., FGFR2 and FGFR3) and proteoglycans (e.g., syndecans [Sdc1, Sdc2, Sdc3], glypicans [Gpc1], versican [Vcan]). Runx2 increases expression of the glycosyltransferase Exostosin-1 (Ext1) and heparanase, as well as alters the relative expression of N-linked sulfotransferases (Ndst1 = Ndst2 > Ndst3) and enzymes mediating O-linked sulfation of heparan sulfate (Hs2st > Hs6st) or chondroitin sulfate (Cs4st > Cs6st). Runx2 cooperates with FGF2 to induce expression of Sdc4 and the sulfatase Galns, but Runx2 and FGF2 suppress Gpc6, thus suggesting intricate Runx2 and FGF2 dependent changes in proteoglycan utilization. One functional consequence of Runx2 mediated modulations in proteoglycan-related gene expression is a change in the responsiveness of bone markers to FGF2 stimulation. Runx2 and FGF2 synergistically enhance osteopontin expression (>100 fold), while FGF2 blocks Runx2 induction of alkaline phosphatase. Our data suggest that Runx2 and the FGF/proteoglycan axis may form an extracellular matrix (ECM)-related regulatory feed-back loop that controls osteoblast proliferation and execution of the osteogenic program.
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PMID:The osteogenic transcription factor Runx2 regulates components of the fibroblast growth factor/proteoglycan signaling axis in osteoblasts. 1925 85

Bone Morphogenetic Proteins (BMPs) are members of the TGF-beta superfamily of growth factors. Several BMPs exhibit osteoinductive bioactivities, and are critical for bone formation in both developing and mature skeletal systems. BMP-7 (OP-1) is currently used clinically in revision of posterolateral spine fusions and long bone non-unions. The current study characterizes BMP-7 induced gene expression during early osteoblastic differentiation of human mesenchymal stem cells (hMSC). Primary hMSC were treated with BMP-7 for 24 or 120 h and gene expression across the entire human genome was evaluated using Affymetrix HG-U133 Plus 2.0 Arrays. 955 probe sets representing 655 genes and 95 ESTs were identified as differentially expressed and were organized into three major expression profiles (Profiles A, B and C) by hierarchical clustering. Genes from each profile were classified according to biochemical pathway analyses. Profile A, representing genes upregulated by BMP-7, revealed strong enrichment for established osteogenic marker genes, as well as several genes with undefined roles in osteoblast function, including MFI2, HAS3, ADAMTS9, HEY1, DIO2 and FGFR3. A functional screen using siRNA suggested roles for MFI2, HEY1 and DIO2 in osteoblastic differentiation of hMSC. Profile B contained genes transiently downregulated by BMP-7, including numerous genes associated with cell cycle regulation. Follow-up studies confirmed that BMP-7 attenuates cell cycle progression and cell proliferation during early osteoblastic differentiation. Profile C, comprised of genes continuously downregulated by BMP-7, exhibited strong enrichment for genes associated with chemokine/cytokine activity. Inhibitory effects of BMP-7 on cytokine secretion were verified by analysis of enriched culture media. Potent downregulation of CHI3L1, a potential biomarker for numerous joint diseases, was also observed in Profile C. A focused evaluation of BMP, GDF and BMP inhibitor expression elucidated feedback loops modulating BMP-7 bioactivity. BMP-7 was found to induce BMP-2 and downregulate GDF5 expression. Transient knockdown of BMP-2 using siRNA demonstrated that osteoinductive properties associated with BMP-7 are independent of endogenous BMP-2 expression. Noggin was identified as the predominant inhibitor induced by BMP-7 treatment. Overall, this study provides new insight into key bioactivities characterizing early BMP-7 mediated osteoblastic differentiation.
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PMID:New insights into BMP-7 mediated osteoblastic differentiation of primary human mesenchymal stem cells. 1930 56

Although substance P (SP) is associated with osteoclast differentiation and bone resorption, little is known about the osteogenic differentiation-inducing effects of SP in periodontal ligament (PDL) cells. This study investigated whether PDL cells could differentiate into osteoblastic-like cells by SP. The expression of osteoblastic differentiation markers such as osteopontin (OPN), osteonectin (ON), osteocalcin (OCN) and bone sialoprotein (BSP) were evaulated by Western blotting. Additionally, SP-mediated heme oxygenase-1 (HO-1) pathways were further clarified. SP increased HO-1 and osteogenic differentiation in concentration- and time-dependent manners, as determined by OPN, ON, OCN and BSP expression. Furthermore, treatment with inhibitors of p38, ERK MAPK, and NF-kappaB abolished SP-induced osteogenic differentiation and HO-1 expression. SP-induced translocation of Nrf-2 was also observed. The combined results suggest that SP activates the stress-response enzymes HO-1 and Nrf-2, subsequently leading to upregulation of osteogenic differentiation in human PDL cells.
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PMID:Effects of substance P on osteoblastic differentiation and heme oxygenase-1 in human periodontal ligament cells. 1935 3


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