Molecular requirements for induction of CTGF expression by TGF-β1 in primary osteoblasts

Autor: S.N. Popoff, John A. Arnott, Fayez F. Safadi, X. Zhang, Steven L. Smock, W. G. Delong, Saqib Rehman, Thomas A. Owen, Archana Sanjay
Rok vydání: 2008
Předmět:
Histology
Physiology
Recombinant Fusion Proteins
Endocrinology
Diabetes and Metabolism
Proto-Oncogene Proteins pp60(c-src)
Response element
Electrophoretic Mobility Shift Assay
Smad Proteins
SMAD
Biology
Response Elements
Article
Immediate early protein
Immediate-Early Proteins
Transforming Growth Factor beta1
Animals
SMAD binding
Src family kinase
Phosphorylation
RNA
Small Interfering
Extracellular Signal-Regulated MAP Kinases
Luciferases
Protein Kinase Inhibitors
Cells
Cultured
Flavonoids
Mitogen-Activated Protein Kinase 1
Mitogen-Activated Protein Kinase 3
Osteoblasts
integumentary system
Connective Tissue Growth Factor
Molecular biology
Rats
CTGF
Pyrimidines
Animals
Newborn
Gene Expression Regulation
Mutagenesis
Site-Directed
Intercellular Signaling Peptides and Proteins
Signal transduction
Protein Binding
Signal Transduction
Proto-oncogene tyrosine-protein kinase Src
Zdroj: Bone. 42:871-885
ISSN: 8756-3282
DOI: 10.1016/j.bone.2008.01.006
Popis: Connective tissue growth factor (CTGF/CCN2) is a cysteine rich, extracellular matrix protein that acts as an anabolic growth factor to regulate osteoblast differentiation and function. In osteoblasts, CTGF is induced by TGF-beta1 where it acts as a downstream mediator of TGF-beta1 induced matrix production. The molecular mechanisms that control CTGF induction by TGF-beta1 in osteoblasts are not known. To assess the role of individual Smads in mediating the induction of CTGF by TGF-beta1, we used specific Smad siRNAs to block Smad expression. These studies demonstrated that Smads 3 and 4, but not Smad 2, are required for TGF-beta1 induced CTGF promoter activity and expression in osteoblasts. Since the activation of MAPKs (Erk, Jnk and p38) by TGF-beta1 is cell type specific, we were interested in determining the role of individual MAPKs in TGF-beta1 induction of CTGF promoter activity and expression. Using dominant negative (DN) mutants for Erk, Jnk and p38, we demonstrated that the expression of DN-Erk caused a significant inhibition of TGF-beta1 induced CTGF promoter activity. In contrast, the expression of DN-p38 or DN-Jnk failed to inhibit activation of CTGF promoter activity. To confirm the vital role of Erk, we used the Erk inhibitor (PD98059) to block its activation, demonstrating that it prevented TGF-beta1 activation of the CTGF promoter and up-regulation of CTGF expression in osteoblasts. Since Src can also act as a downstream signaling effector for TGF-beta in some cell types, we determined its role in TGF-beta1 induction of CTGF in osteoblasts. Treatment of osteoblasts with a Src family kinase inhibitor, PP2, or the expression of two independent kinase-dead Src mutant constructs caused significant inhibition of TGF-beta1 induced CTGF promoter activity and expression. Additionally, blocking Src activation prevented Erk activation by TGF-beta1 demonstrating a role for Src as an upstream mediator of Erk in regulating CTGF expression in osteoblasts. To investigate the involvement of the TGF-beta1 response element (TRE) and the SMAD binding element (SBE) in CTGF induction, we cloned the rat CTGF proximal promoter (-787 to +1) containing the TRE and SBE motifs into a pGL3-Luciferase reporter construct. Using a combination of CTGF promoter deletion constructs and site-directed mutants, we demonstrated the unique requirement of both the TRE and SBE for CTGF induction by TGF-beta1 in osteoblasts. Electro-mobility shift assays using specific probes containing the TRE, SBE or both showed TGF-beta1 inducible complexes that can be ablated by mutation of the respective motif, confirming their requirement for TGF-beta1 induced CTGF promoter activity. In conclusion, these studies demonstrate that CTGF induction by TGF-beta1 in osteoblasts involves Smads 3 and 4, the Erk and Src signaling pathways, and requires both the TRE and SBE motifs in the CTGF proximal promoter.
Databáze: OpenAIRE
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