Retinoic acid selectively activates the ERK2 but not JNK/SAPK or P38 map kinases when inducing myeloid differentiation

Summary Among the three major mitogen-activated protein kinase (MAPK) cascades—the extracellular signal regulated kinase (ERK) pathway, the c-JUN N-terminal/stress-activated protein kinase (JNK/SAPK) pathway, and the reactivating kinase (p38) pathway—retinoic acid selectively utilizes ERK but not JNK/SAPK or p38 when inducing myeloid differentiation of HL-60 human myeloblastic leukemia cells. Retinoic acid is known to active ERK2. The present data show that the activation is selective for this MAPK pathway. JNK/SAPK or p38 are not activated by retinoic acid. Presumably because it activates relevant signaling pathways including MAPK, the polyoma middle T antigen, as well as certain transformation defective mutants thereof, is known to promote retinoic acid-induced differentiation, although the mechanism of action is not well understood. The present results show that consistent with the selective involvement of ERK2, ectopic expression of either the polyoma middle T antigen or its dl23 mutant, which is defective for PLCγ and PI-3 kinase activation, or the Δ205 mutant, which in addition is also weakened for activation of src-like kinases, caused no enhanced JNK/SAPK or p38 kinase activity that promoted the effects of retinoic acid. However, all three of these polyoma antigens are known to enhance ERK2 activation and promote differentiation induced by retinoic acid. Polyoma-activated MAPK signaling relevant to retinoic acid-induced differentiation is thus restricted to ERK2 and does not involve JNK/SAPK or p38. Taken together, the data indicate that among the three parallel MAPK pathways, retinoic acid-induced HL-60 myeloid differentiation selectively depends on activating ERK but not the other two MAPK pathways, JNK/SAPK or p38, with no apparent cross talk between pathways. Furthermore, the striking ability of polyoma middle T antigens to promote retinoic acid-induced differentiation appears to utilize ERK, but not JNK/SPK or p38 signaling.     

ATF1 phosphorylation by the ERK MAPK pathway is required for epidermal growth factor-induced c-jun expression

Epidermal growth factor induction of c-jun expression requires ATF1 and MEF2 sites in the c-jun promoter. We find that activation of the c-jun promoter through the ATF1 site requires phosphorylation of ATF1 at serine 63. A serine 63 to alanine mutation of ATF1 acts to block epidermal growth factor (EGF) induction of a transfected c-jun gene. ATF1 can be phosphorylated by mitogen- and stress-activated protein kinase 1 (MSK1), which is activated by EGF and ERK1/2. Kinase-dead MSK1 mutants blocked EGF induction of a transfected c-jun gene suggesting that MSK1 or a similar family member is required for induced c-jun expression. Use of the MEK1 inhibitor U0126 and dominant negative MEK1 further showed that MSK1 activation and c-jun induction require the ERK pathway. In contrast, a JNK inhibitor blocked EGF induction of c-jun expression but not ATF1 phosphorylation. These results show that the two MAPK pathways, ERK and JNK, are required for EGF-induced c-jun expression and that the ERK pathway acts through downstream phosphorylation of ATF1.     

The kinase domain of MEKK1 induces apoptosis by dysregulation of MAP kinase pathways

MAP kinase pathways comprise a group of parallel protein phosphorylation cascades, which are involved in signaling triggered by a variety of stimuli. Previous findings suggested that the ERK and the JNK pathways have opposing roles in regulating proliferation and survival or apoptosis and that apoptosis can be promoted by inhibiting the ERK pathway or by activation of the JNK pathway. In order to test this hypothesis and explore whether it can be exploited as a strategy for killing human cancer cells, we used gene transfer experiments with a range of cancer cell lines. We expressed the catalytic fragment of human MEKK1 to activate JNK and the Ras-binding domain (RBD) of Raf-1 to inhibit the Ras-ERK pathway. In addition, we designed several RBD-MEKK1 fusion proteins aiming to simultaneously activate the JNK and block the ERK pathway. We found that the MEKK1 proteins as well as the RBD alone could reduce colony formation in all cell lines. The survival time of MEKK1-expressing cells depended on the cell line. In HeLa cells, survival could be prolonged by inhibition of caspases but not by coexpression of the anti-apoptotic protein Bcl-2. Due to a lower kinase activity the RBD-MEKK1 fusion proteins were less effective in apoptosis induction than the MEKK1 kinase domain alone. Using mutant forms of Ras and Raf-1 we could show that the reduced kinase activity of RBD-MEKK1 fusion proteins was caused by binding to the Ras protein. The expression of lethal doses of MEKK1 resulted in a strong activation of all three major MAP kinase families JNK, ERK, and p38. Blocking these pathways either by coexpressing a dominant negative form of MKK4 or with inhibitors of MEK or p38 failed to inhibit apoptosis. This suggests that MEKK1 induces apoptosis by causing a general deregulation of MAP kinase signaling rather than by the activation of a single pathway.     

Systemic transplantation of human adipose-derived stem cells stimulates bone repair by promoting osteoblast and osteoclast function

Systemic transplantation of adipose-derived stem cells (ASCs) is emerging as a novel therapeutic option for functional recovery of diverse damaged tissues. This study investigated the effects of systemic transplantation of human ASCs (hASCs) on bone repair. We found that hASCs secrete various bone cell-activating factors, including hepatocyte growth factor and extracellular matrix proteins. Systemic transplantation of hASCs into ovariectomized mice induced an increased number of both osteoblasts and osteoclasts in bone tissue and thereby prevented bone loss. We also observed that conditioned medium from hASCs is capable of stimulating proliferation and differentiation of osteoblasts via Smad/extracellular signal-regulated kinase (ERK)/JNK (c-jun NH(2) -terminal kinase) activation as well as survival and differentiation of osteoclasts via ERK/JNK/p38 activation in vitro. Overall, our findings suggest that paracrine factors secreted from hASCs improve bone repair and that hASCs can be a valuable tool for use in osteoporosis therapy.     

Angiotensin II induces the production of MMP-3 and MMP-13 through the MAPK signaling pathways via the AT1 receptor in osteoblasts

Angiotensin II (Ang II) plays an important role in the maintenance of bone mass and integrity by activation of the mitogen-activated protein kinases (MAPKs) and by modulation of balance between resorption by osteoclasts and formation by osteoblasts. However, the role of Ang II in the turnover of extracellular matrix (ECM) in osteoid by osteoblasts remains unclear. Therefore, we examined the effect of Ang II on the expression of matrix metalloproteinases (MMPs), plasminogen activators (PAs), and their inhibitors [i.e., tissue inhibitors of metalloproteinases (TIMPs) and PA inhibitor-1 (PAI-1)] using osteoblastic ROS17/2.8 cells. Treatment with Ang II strikingly increased the expressions of MMP-3 and -13 and promoted cell proliferation associated with reduced alkaline phosphatase activity as well as enhanced phosphorylated expression of extracellular signal-regulated kinase (ERK)1/2, p38 MAPK, and stress-activated protein kinases/c-jun N-terminal kinases (SAPK/JNK) in ROS17/2.8 cells. However, Ang II had no effect on the expression of MMP-2, -9, -14, urokinase-type PA, tissue-type PA, TIMP-1, -2, -3, and PAI-1 in cells. Losartan (AT₁ receptor blocker) blocked Ang II-induced expression of MMP-3 and -13, whereas PD123319 (AT₂ receptor blocker) did not completely block these responses. Losartan also blocked the Ang II-induced phosphorylation of ERK1/2, p38 MAPK, and SAPK/JNK. MAPK kinase 1/2 inhibitor PD98059 and JNK inhibitor SP600125 suppressed Ang II-induced expression of MMP-3 and -13. These results suggested that Ang II stimulated the degradation process that occurs during ECM turnover in osteoid by increasing the production of MMP-3 and -13 through MAPK signaling pathways via the AT₁ receptor in osteoblasts. Furthermore, our findings suggest that Ang II does not influence the plasminogen/plasmin pathway in osteoblasts.     

CYR61 Regulates BMP-2-dependent Osteoblast Differentiation through the αvβ3 Integrin/Integrin-linked Kinase/ERK Pathway

Osteoporosis is one of the most common bone pathologies. A number of novel molecules have been reported to increase bone formation including cysteine-rich protein 61 (CYR61), a ligand of integrin receptor, but mechanisms remain unclear. It is known that bone morphogenetic proteins (BMPs), especially BMP-2, are crucial regulators of osteogenesis. However, the interaction between CYR61 and BMP-2 is unclear. We found that CYR61 significantly increases proliferation and osteoblastic differentiation in MC3T3-E1 osteoblasts and primary cultured osteoblasts. CYR61 enhances mRNA and protein expression of BMP-2 in a time- and dose-dependent manner. Moreover, CYR61-mediated proliferation and osteoblastic differentiation are significantly decreased by knockdown of BMP-2 expression or inhibition of BMP-2 activity. In this study we found integrin α_vβ₃ is critical for CYR61-mediated BMP-2 expression and osteoblastic differentiation. We also found that integrin-linked kinase, which is downstream of the α_vβ₃ receptor, is involved in CYR61-induced BMP-2 expression and subsequent osteoblastic differentiation through an ERK-dependent pathway. Taken together, our results show that CYR61 up-regulates BMP-2 mRNA and protein expression, resulting in enhanced cell proliferation and osteoblastic differentiation through activation of the α_vβ₃ integrin/integrin-linked kinase/ERK signaling pathway.     

Connective tissue growth factor is a downstream mediator for preptin-induced proliferation and differentiation in human osteoblasts

Preptin, a newly isolated 34-amino-acid peptide hormone that is cosecreted with insulin and amylin from pancreatic beta-cells, has emerged as a regulatory element in bone metabolism, but its mechanism remains unclear. We assessed the effects of preptin on proliferation and differentiation of human osteoblasts and investigated the mechanism involved. Our results demonstrated that preptin promoted human osteoblasts proliferation and alkaline phosphatase activity. Suppression of connective tissue growth factor (CTGF), which was upregulated by preptin in a dose- and time-dependent manner, with small interfering RNA (siRNA) abolished the preptin-induced human osteoblasts proliferation and differentiation. Preptin induced activation of ERK mitogen-activated protein kinase (MAPK), but not p38 or JNK in human osteoblasts. Furthermore, pretreatment of human osteoblasts with the ERK inhibitor PD98059 abolished the preptin-induced CTGF secretion and blocked the promoting effect of preptin on osteoblasts proliferation and differentiation. These data demonstrated that preptin is involved in bone anabolism mediated by ERK/CTGF in human osteoblasts and may contribute to the preservation of bone mass observed in hyperinsulinemic states, such as obesity.     

Taurine promotes connective tissue growth factor (CTGF) expression in osteoblasts through the ERK signal pathway

Summary. Taurine is found in bone tissue, but its function in skeletal tissue is not fully understood. The present study was undertaken to investigate regulation of gene expression of connective tissue growth factor (CTGF), and the roles of mitogen-activated protein kinases (MAPKs) in murine osteoblast MC3T3-E1 cells treated with taurine. Western blot analysis showed taurine stimulated CTGF protein secretion in a dose- and time-dependent manner. Taurine induced activation of extracellular signal-regulated kinase (ERK), but not p38 and c-jun N-terminal Kinase (JNK), in osteoblasts. Furthermore, pretreatment of osteoblasts with the ERK inhibitor PD98059 abolished the taurine-induced CTGF production. These data indicate that taurine induces CTGF secretion in MC3T3-E1 cells mediated by the ERK pathway, and suggest that osteoblasts are direct targets of taurine.     

Adult human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is regulated by mitogen-activated protein kinase

Adult human mesenchymal stem cells are primary, multipotent cells capable of differentiating to osteocytic, chondrocytic, and adipocytic lineages when stimulated under appropriate conditions. To characterize the molecular mechanisms that regulate osteogenic differentiation, we examined the contribution of mitogen-activated protein kinase family members, ERK, JNK, and p38. Treatment of these stem cells with osteogenic supplements resulted in a sustained phase of ERK activation from day 7 to day 11 that coincided with differentiation, before decreasing to basal levels. Activation of JNK occurred much later (day 13 to day 17) in the osteogenic differentiation process. This JNK activation was associated with extracellular matrix synthesis and increased calcium deposition, the two hallmarks of bone formation. Inhibition of ERK activation by PD98059, a specific inhibitor of the ERK signaling pathway, blocked the osteogenic differentiation in a dose-dependent manner, as did transfection with a dominant negative form of MAP kinase kinase (MEK-1). Significantly, the blockage of osteogenic differentiation resulted in the adipogenic differentiation of the stem cells and the expression of adipose-specific mRNAs peroxisome proliferator-activated receptor gamma2, aP2, and lipoprotein lipase. These observations provide a potential mechanism involving MAP kinase activation in osteogenic differentiation of adult stem cells and suggest that commitment of hMSCs into osteogenic or adipogenic lineages is governed by activation or inhibition of ERK, respectively.