Abstract
Transforming growth factor (TGF) beta inhibits alkaline phosphatase (ALP) activity and mineralization in mouse osteoblastic MC3T3-E1 cells, whereas local administration of TGF-beta stimulates bone formation in vivo. We recently demonstrated that Smad3, a TGF-beta signaling molecule, promotes ALP activity and mineralization in MC3T3-E1 cells. Moreover, the target disruption of Smad3 in mouse is reported to cause a decrease in bone mineral density. These findings indicate that Smad3 plays an important role in the regulation of bone formation. However, why the effects of TGF-beta and Smad3 on ALP activity and mineralization are different remains unknown. The purpose of the present study is to clarify the role of mitogen-activated protein kinase (MAPK) in TGF-beta and Smad3 pathways in osteoblast. TGF-beta activated extracellular signal-regulated kinases/p42/p44 (ERK1/2), p38 MAPK, and c-Jun N-terminal kinase (JNK) in mouse osteoblastic MC3T3-E1 cells. The expression of dominant negative type Smad3, Smad3DeltaC, affected neither TGF-beta-activated MAPKs nor TGF-beta-inhibited ALP activity. Specific inhibitors of ERK1/2 activation (PD98059 and U0126), as well as JNK inhibitors (curcumin and dicumarol) antagonized the inhibitory effects of TGF-beta on ALP activity and mineralization, whereas the specific inhibitor of p38 MAPK (SB203580) did not affect them. PD98059 and curcumin enhanced Smad3-induced ALP activity and mineralization, whereas SB203580 inhibited them. In the luciferase reporter assay using 3TP-lux with the specific Smad3-responsive element, PD98059, and curcumin enhanced TGF-beta- and Smad3-induced transcriptional activity in MC3T3-E1 cells. On the other hand, TGF-beta-induced production of type I collagen was antagonized by curcumin but not by PD98059. The present study indicated that TGF-beta-responsive ERK1/2 and JNK cascades negatively regulate Smad3-induced transcriptional activity as well as ALP activity and mineralization in osteoblasts.
Highlights
Transforming growth factor  (TGF-)1 is most abundant in bone matrix compared with other tissues [1]
These inhibitors did not affect the basal levels of total ERK1/2, p38 MAPK (P38), and Jun N-terminal kinase (JNK). These specific inhibitors did not affect the phosphorylation of other mitogen-activated protein kinase (MAPK); for example, PD98059 could not inhibit TGF--induced phosphorylation of P38 and JNK. These results indicated that the inhibitors of MAPKs employed in the present study antagonized TGF--induced activation of ERK1/2, P38, and JNK in MC3T3-E1 cells
The inhibition of the Smad3 signaling pathway by the expression of dominant negative type Smad3, Smad3⌬C, did not affect TGF--induced activations of MAPKs, ERK1/2, P38, and JNK. These findings indicated that activations of these MAPKs by TGF- were independent of Smad3 signaling pathways
Summary
Transforming growth factor  (TGF-) is most abundant in bone matrix compared with other tissues [1]. Alliston et al [15] reported that Smad decreased Runx and osteocalcin gene expressions in MC3T3-E1 cells, our recent study revealed that Smad inhibited the proliferation and enhanced the levels of bone matrix proteins, such as type I collagen, osteopontin, and matrix Gla protein in a manner similar to TGF- in these cells [16]. There are three distinct MAPKs that have been identified in mammalian cells, referred to as extracellular signal-regulated kinases/p42/44 MAPK (ERK1/2), p38 MAPK (P38), and c-Jun N-terminal kinases (JNK)/stress-activated protein kinases [18]. These MAPKs are all proline-directed, This paper is available on line at http://www.jbc.org. Our aim of this study is to clarify the role of MAPKs in TGF- and Smad pathways in osteoblastic cells
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