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Activations of ERK1/2 and JNK by transforming growth factor beta negatively regulate Smad3-induced alkaline

Hideaki Sowa1, Hiroshi Kaji, Toru Yamaguchi

  • 1Division of Endocrinology/Metabolism, Neurology and Hematology/Oncology, Department of Clinical Molecular Medicine, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-Cho, Chuo-ku, Kobe 650-0017, Japan.

The Journal of Biological Chemistry
|July 20, 2002
PubMed

Insights

Transforming growth factor-beta (TGF-β) signaling in osteoblasts involves mitogen-activated protein kinases (MAPKs). ERK1/2 and JNK pathways negatively regulate Smad3

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Bone Biology
  • Signaling Pathways

Background:

  • Transforming growth factor-beta (TGF-β) exhibits context-dependent effects on bone formation, inhibiting alkaline phosphatase (ALP) activity and mineralization in osteoblastic cells but stimulating bone formation in vivo.
  • Smad3, a key TGF-β signaling molecule, promotes ALP activity and mineralization in osteoblasts, and its deficiency in mice leads to reduced bone mineral density, highlighting its crucial role in bone formation.
  • The differential effects of TGF-β and Smad3 on osteoblast activity remain unclear, prompting an investigation into the involvement of mitogen-activated protein kinases (MAPKs) in these pathways.

Purpose of the Study:

  • To elucidate the role of mitogen-activated protein kinase (MAPK) signaling in mediating the opposing effects of Transforming growth factor-beta (TGF-β) and Smad3 on osteoblast differentiation.
  • To investigate how TGF-β-activated MAPKs interact with the Smad3 signaling pathway in regulating alkaline phosphatase (ALP) activity and mineralization in mouse osteoblastic MC3T3-E1 cells.

Main Methods:

  • Treatment of mouse osteoblastic MC3T3-E1 cells with TGF-β and specific MAPK inhibitors (PD98059, U0126 for ERK1/2; curcumin, dicumarol for JNK; SB203580 for p38 MAPK).
  • Assessment of alkaline phosphatase (ALP) activity and mineralization.
  • Luciferase reporter assays using a Smad3-responsive element (3TP-lux) to evaluate transcriptional activity.
  • Analysis of type I collagen production.

Main Results:

  • TGF-β activated extracellular signal-regulated kinases/p42/p44 (ERK1/2), p38 MAPK, and c-Jun N-terminal kinase (JNK) in MC3T3-E1 cells.
  • Inhibition of ERK1/2 and JNK, but not p38 MAPK, antagonized TGF-β's inhibitory effects on ALP activity and mineralization.
  • ERK1/2 and JNK inhibition enhanced Smad3-induced ALP activity and mineralization, and boosted TGF-β- and Smad3-mediated transcriptional activity, while p38 MAPK inhibition had opposing effects.
  • TGF-β-induced type I collagen production was inhibited by JNK inhibition (curcumin) but not by ERK1/2 inhibition (PD98059).

Conclusions:

  • TGF-β-responsive ERK1/2 and JNK signaling cascades act as negative regulators of Smad3-induced transcriptional activity, alkaline phosphatase (ALP) activity, and mineralization in osteoblasts.
  • These findings clarify the complex interplay between TGF-β, Smad3, and MAPK pathways in osteoblast differentiation and provide insights into the regulation of bone formation.

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