AMP-activated protein kinase (AMPK) is an energy-sensing kinase that has recently been shown to regulate the differentiation of preadipocytes and osteoblasts. However, the role of AMPK in stem cell differentiation is largely unknown. Using in vitro culture models, the present study demonstrates that AMPK is a critical regulatory factor for osteogenic differentiation. We observed that expression and phosphorylation of AMPK were increased during osteogenesis in human adipose tissue-derived mesenchymal stem cells (hAMSC). To elucidate the role of AMPK in osteogenic differentiation, we investigated the effect of AMPK inhibition or knockdown on mineralization of hAMSC. Compound C, an AMPK inhibitor, reduced mineralized matrix deposition and suppressed the expression of osteoblast-specific genes, including alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN). Knockdown of AMPK by shRNA-lentivirus infection also reduced osteogenesis. In addition, inhibition or knockdown of AMPK during osteogenesis inhibited ERK phosphorylation, which is required for osteogenesis. Interestingly, inhibition of AMPK induced adipogenic differentiation of hAMSC, even in osteogenic induction medium (OIM). These results provide a potential mechanism involving AMPK activation in osteogenic differentiation of hAMSC and suggest that commitment of hAMSC to osteogenic or adipogenic lineage is governed by activation or inhibition of AMPK, respectively.

The lymphatic system plays pivotal roles in mediating tissue fluid homeostasis and immunity, and excessive lymphatic vessel formation is implicated in many pathological conditions, which include inflammation and tumor metastasis. However, the molecular mechanisms that regulate lymphatic vessel formation remain poorly characterized. Sphingosine-1-phosphate (S1P) is a potent bioactive lipid that is implicated in a variety of biologic processes such as inflammatory responses and angiogenesis. Here, we first report that S1P acts as a lymphangiogenic mediator. S1P induced migration, capillarylike tube formation, and intracellular Ca 2؉ mobilization, but not proliferation, in human lymphatic endothelial cells (HLECs) in vitro. Moreover, a Matrigel plug assay demonstrated that S1P promoted the outgrowth of new lymphatic vessels in vivo. HLECs expressed S1P1 and S1P3, and both RNA interference-mediated down-regulation of S1P1 and an S1P1 antagonist significantly blocked S1P-mediated lymphangiogenesis. Furthermore, pertussis toxin, U73122, and BAPTA-AM efficiently blocked S1P-induced in vitro lymphangiogenesis and intracellular Ca 2؉ mobilization of HLECs, indicating that S1P promotes lymphangiogenesis by stimulating S1P1/G i /phospholipase C/Ca 2؉ signaling pathways. Our results suggest that S1P is the first lymphangiogenic bioactive lipid to be identified, and that S1P and its receptors might serve as new therapeutic targets against inflammatory diseases and lymphatic metastasis in tumors. (Blood. 2008; 112:1129-1138) IntroductionSphingosine-1-phosphate (S1P) has been implicated in a wide spectrum of biologic processes, including the promotion of cell growth and survival, migration and differentiation, platelet aggregation, inflammatory responses, and angiogenesis. 1 S1P is generated by the phosphorylation of sphingosine through a process mediated by sphingosine kinase 1 (SphK1) and SphK2. S1P acts both intracellularly as a second messenger 2 and extracellularly as a ligand for a family of G-protein-coupled S1P receptors. 3 S1P1 couples stringently to the G i protein family, whereas S1P2 and S1P3 couple to the G i , G q , and G 12/13 protein families. Multiple interconnections of S1P signaling through S1P1 and S1P3 induce vascular endothelial cell proliferation, migration, morphogenesis, cytoskeletal reorganization, and adherens junction assembly, whereas signaling via S1P2 negatively regulates S1P-mediated multiple responses of vascular endothelial cells. 4 The defective vascular maturation observed in S1P1-deficient mice highlights a fundamental role for S1P signaling on vasculogenesis. 5 Neutralization of the action of extracellular S1P shows significant inhibition of angiogenesis, tumor growth, metastasis, and lymphocyte transmigration, indicating that S1P is an important pathological regulator of inflammation and angiogenesis. [6][7][8] Lymphatic vessels play important roles in mediating tissue fluid homeostasis and immunity. 9 Although lymphangiogenesis, the formation of new lymphatic vessels from preexisting ves...

Adipogenesis is a complex process that is accompanied by a number of molecular events. In this study, a proteomic approach was adopted to identify secretory factors associated with adipogenesis. A label-free shotgun proteomic strategy was implemented to analyze proteins secreted by human adipose stromal vascular fraction cells and differentiated adipocytes. A total of 474 proteins were finally identified and classified according to quantitative changes and statistical significances. Briefly, 177 proteins were significantly upregulated during adipogenesis (Class I), whereas 60 proteins were significantly downregulated (Class II). Changes in the expressions of several proteins were confirmed by quantitative RT-PCR and immunoblotting. One obvious finding based on proteomic data was that the amounts of several extracellular modulators of Wnt and transforming growth factor-beta (TGF-beta) signaling changed during adipogenesis. The expressions of secreted frizzled-related proteins, dickkopf-related proteins, and latent TGF-beta-binding proteins were found to be altered during adipogenesis, which suggests that they participate in the fine regulation of Wnt and TGF-beta signaling. This study provides useful tools and important clues regarding the roles of secretory factors during adipogenic differentiation, and provides information related to obesity and obesity-related metabolic diseases.

Communication between osteoblasts and endothelial cells is essential for bone fracture repair, but the molecular identities of such communicating factors are not well defined. Here we identify DJ-1 as a novel mediator of the cross-talk between osteoblasts and endothelial cells through an unbiased screening of molecules secreted from human mesenchymal stem cells during osteogenesis. We show that DJ-1 stimulates the differentiation of human mesenchymal stem cells to osteoblasts and that DJ-1 induces angiogenesis in endothelial cells through activation of fibroblast growth factor receptor-1 signalling. In a rodent model of bone fracture repair, extracellular application of DJ-1 enhances bone regeneration in vivo by stimulating the formation of blood vessels and new bones. Both these effects are blocked by antagonizing fibroblast growth factor receptor-1 signalling. These findings uncover previously undefined extracellular roles of DJ-1 to promote angiogenesis and osteogenesis, suggesting DJ-1 may have therapeutic potential to stimulate bone regeneration.

Lysophosphatidic acid (LPA) is a bioactive phospholipid that affects various biological functions, such as cell proliferation, migration, and survival, through LPA receptors. Among them, the motility of cancer cells is an especially important activity for invasion and metastasis. Recently, AMP-activated protein kinase (AMPK), an energy-sensing kinase, was shown to regulate cell migration. However, the specific role of AMPK in cancer cell migration is unknown. The present study investigated whether LPA could induce AMPK activation and whether this process was associated with cell migration in ovarian cancer cells. We found that LPA led to a striking increase in AMPK phosphorylation in pathways involving the phospholipase C-␤3 (PLC-␤3) and calcium/calmodulin-dependent protein kinase kinase ␤ (CaMKK␤) in SKOV3 ovarian cancer cells. siRNA-mediated knockdown of AMPK␣1, PLC-␤3, or (CaMKK␤) impaired the stimulatory effects of LPA on cell migration. Furthermore, we found that knockdown of AMPK␣1 abrogated LPA-induced activation of the small GTPase RhoA and ezrin/radixin/moesin proteins regulating membrane dynamics as membrane-cytoskeleton linkers. In ovarian cancer xenograft models, knockdown of AMPK significantly decreased peritoneal dissemination and lung metastasis. Taken together, our results suggest that activation of AMPK by LPA induces cell migration through the signaling pathway to cytoskeletal dynamics and increases tumor metastasis in ovarian cancer. Lysophosphatidic acid (LPA)2 has been shown to participate in diverse biological actions, including changes in cell shape, motility, and proliferation, in a variety of cell types (1). Previous studies have shown that LPA has a role in early signaling events, such as Ca 2ϩ mobilization, changes in cAMP accumulation, and the activation of several protein kinases (1-4). Among these pathological processes, the role of LPA in ovarian cancer has been most extensively studied. LPA contributes to the development, progression, and metastasis of ovarian cancer, and its concentration is increased up to 80 M (in comparison to the basal 1-5 M concentration) in both plasma and ascites of ovarian cancer patients (5). In vitro studies have shown that production of LPA levels was constitutively increased in ovarian cancer cells but not in normal ovarian epithelial cells (6, 7). Moreover, in a study of the expression of LPA receptor mRNA and protein levels in ovarian cancer tissues, LPA 2 and LPA 3 were aberrantly up-regulated, but LPA 1 was not changed (8, 9). Overexpression of LPA 2 and LPA 3 are closely associated with tumor progression in ovarian cancer cells (10 -13). As evidence of intracellular signaling in cancer cell migration, LPA induces activation of Ras-MEKK1 (14), Rac1 (15), Ca 2ϩ -dependent Pyk2 (16), and the Rho/ROCK pathway (17), which indicates that dynamic cytoskeletal rearrangement in LPA-mediated cell migration is regulated through the coordination of complex contexts (such as small GTPases, focal adhesion, and Ca 2ϩ -dependent signaling). However, the exact re...