Characterization of Runx2 phosphorylation sites required for TGF-β1-mediated stimulation of matrix metalloproteinase-13 expression in osteoblastic cells

Balasubramanian Arumugam, Mariappanadar Vairamani, Nicola Partridge, Nagarajan Selvamurugan

Research output: Contribution to journalArticle

Abstract

Transforming growth factor-beta1 (TGF-β1), a highly abundant growth factor in skeletal tissues, stimulates matrix metalloproteinase-13 (MMP-13) expression in osteoblastic cells. MMP-13 plays a critical role in bone remodeling. Runx2, a bone transcription factor, is required for TGF-β1-mediated stimulation of MMP-13 expression in osteoblastic cells. In this study, the molecular mechanism responsible for TGF-β1-stimulation of MMP-13 expression via Runx2 in osteoblastic cells was elucidated. TGF-β1 stimulated the phosphorylation of Runx2 at serine amino acids, and ERK inhibition blocked this effect in rat (UMR106-01) and human (MG-63) osteoblastic cells. Pretreatment with okadaic acid, a serine-threonine phosphatase inhibitor, increased Runx2 serine phosphorylation in osteoblastic cells. When cells were pretreated with an ERK inhibitor, TGF-β1-mediated stimulation of MMP-13 mRNA expression decreased. Nano-ESI/LC/MS analysis identified that TGF-β1 stimulates Runx2 phosphorylation at three serine amino acids. Transient transfection of mouse mesenchymal stem cells (C3H10T1/2) with Runx2 serine mutant constructs decreased TGF-β1-mediated Runx2 serine phosphorylation. A luciferase reporter assay identified that TGF-β1 stimulated MMP-13 promoter activity in these cells only in the presence of the wild Runx2 construct, and not with mutant Runx2. Thus, TGF-β1 stimulates the phosphorylation of Runx2 at three serine amino acids, and this event is required for MMP-13 expression in osteoblastic cells. Hence, this study contributes to the knowledge of events governing bone remodeling and bone-related diseases.

Original languageEnglish (US)
JournalJournal of Cellular Physiology
DOIs
StateAccepted/In press - 2017

Fingerprint

Matrix Metalloproteinase 13
Transforming Growth Factor beta1
Phosphorylation
Serine
Bone
Bone Remodeling
Amino Acids
Bone and Bones
Okadaic Acid
Phosphoprotein Phosphatases
Bone Diseases
Stem cells
Luciferases
Mesenchymal Stromal Cells
Transfection
Rats
Assays
Intercellular Signaling Peptides and Proteins
Transcription Factors
Tissue

Keywords

  • MMP-13
  • Phosphorylation
  • TGF-β1 Runx2

ASJC Scopus subject areas

  • Medicine(all)
  • Physiology
  • Clinical Biochemistry
  • Cell Biology

Cite this

Characterization of Runx2 phosphorylation sites required for TGF-β1-mediated stimulation of matrix metalloproteinase-13 expression in osteoblastic cells. / Arumugam, Balasubramanian; Vairamani, Mariappanadar; Partridge, Nicola; Selvamurugan, Nagarajan.

In: Journal of Cellular Physiology, 2017.

Research output: Contribution to journalArticle

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AB - Transforming growth factor-beta1 (TGF-β1), a highly abundant growth factor in skeletal tissues, stimulates matrix metalloproteinase-13 (MMP-13) expression in osteoblastic cells. MMP-13 plays a critical role in bone remodeling. Runx2, a bone transcription factor, is required for TGF-β1-mediated stimulation of MMP-13 expression in osteoblastic cells. In this study, the molecular mechanism responsible for TGF-β1-stimulation of MMP-13 expression via Runx2 in osteoblastic cells was elucidated. TGF-β1 stimulated the phosphorylation of Runx2 at serine amino acids, and ERK inhibition blocked this effect in rat (UMR106-01) and human (MG-63) osteoblastic cells. Pretreatment with okadaic acid, a serine-threonine phosphatase inhibitor, increased Runx2 serine phosphorylation in osteoblastic cells. When cells were pretreated with an ERK inhibitor, TGF-β1-mediated stimulation of MMP-13 mRNA expression decreased. Nano-ESI/LC/MS analysis identified that TGF-β1 stimulates Runx2 phosphorylation at three serine amino acids. Transient transfection of mouse mesenchymal stem cells (C3H10T1/2) with Runx2 serine mutant constructs decreased TGF-β1-mediated Runx2 serine phosphorylation. A luciferase reporter assay identified that TGF-β1 stimulated MMP-13 promoter activity in these cells only in the presence of the wild Runx2 construct, and not with mutant Runx2. Thus, TGF-β1 stimulates the phosphorylation of Runx2 at three serine amino acids, and this event is required for MMP-13 expression in osteoblastic cells. Hence, this study contributes to the knowledge of events governing bone remodeling and bone-related diseases.

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