Abstract
Bone morphogenetic protein (BMP)-2 has the capacity to induce the neuronal differentiation of PC12 cells. Unlike nerve growth factor, however, BMP-2 failed to induce the activation of the 41-/43-kDa mitogen-activated protein (MAP) kinase pathway in these cells. In contrast, BMP-2 characteristically induced the sustained activation of the p38 MAP kinase pathway. Pretreatment of PC12 cells with SB203580 inhibited the BMP-2-induced neurite outgrowth formation in a dose-dependent manner; this inhibition coincided well with the ability of SB203580 to inihibit the BMP-2-induced activation of the p38 MAP kinase pathway. Overexpression in PC12 cells of wild-type MAP kinase kinase (MKK)-6 enhanced the BMP-2-induced activation of p38 MAP kinase, whose activation correlated well with the ability of these cells to induce neurite outgrowth in response to BMP-2. Transient expression of kinase-negative forms of MKK3/6 inhibited the formation of neurite outgrowth in response to BMP-2. Furthermore, expression of constitutively active forms of MKK3/6 induced neurite outgrowth without BMP-2 stimulation, and SB203580 inhibited this induction. These results clearly indicate that activation of the p38 MAP kinase pathway is necessary for BMP-2-induced neuronal differentiation of PC12 cells. Our results also suggest that activation of the p38 MAP kinase pathway alone can induce the neuronal differentiation of PC12 cells.
Highlights
Bone morphogenetic protein (BMP)-2 has the capacity to induce the neuronal differentiation of PC12 cells
We have investigated the possible involvement of other members of the mitogen-activated protein (MAP) kinase family, p38 MAP kinase and JNK, in the BMP-2 signaling pathway that leads to the induction of neuronal differentiation of PC12 cells
We examined the possible involvement of other members of the MAP kinase family, p38 MAP kinase and JNK, in the signaling pathway of BMP-2 and activin A that leads to the induction of neuronal differentiation of PC12 cells
Summary
Materials—Recombinant human BMP-2, expressed in silkworm larvae, was purified to homogeneity as described previously [29]. Davis (University of Massachusetts Medical School), that for wild-type MKK6 (pME-HA-MKK6) [35] was kindly provided by Dr Masatoshi Hagiwara (Tokyo Medical and Dental University), and those for a kinase-negative form of MKK6 (pCS-Myc-MKK6 (Ala); ATP-binding site lysine was replaced with alanine) and a constitutively active form of MKK6 (pCSMyc-MKK6 (Glu); phosphorylation sites Ser-207 and Thr-211 were replaced with glutamic acid) were kindly provided by Dr Kenji Sugiyama (Nippon Boehringer Ingelheim Co., Ltd).2 Transfection of these plasmids into PC12 cells was done by the LipofectAMINE method according to the manufacturer’s instructions (Life Technologies, Inc.), using 5 g of DNA/60-mm dish unless otherwise indicated. For assays of p38 MAP kinase and JNK, reactions were terminated by adding 10 l of 4ϫ SDS sample buffer, and the incorporation of 32P into substrate proteins was examined by SDSpolyacrylamide gel electrophoresis followed by autoradiography using a Fujix Bioimaging analyzer BAS 1500 (Fuji Photo Film Co., Tokyo, Japan). Western Blot Analysis—Cell lysates were separated by SDS-polyacrylamide gel electrophoresis, electrophoretically transferred to an Immobilon-P membrane (Millipore Corp.), and subjected to immunode-
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