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(Circulation Research. 2008;102:e38.)
© 2008 American Heart Association, Inc.

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Angiotensin II–Inducible Platelet-Derived Growth Factor-D Transcription Requires Specific Ser/Thr Residues in the Second Zinc Finger Region of Sp1

Nicole Y. Tan, Valerie C. Midgley, Mary M. Kavurma, Fernando S. Santiago, Xiao Luo, Ryan Peden, Roger G. Fahmy, Michael C. Berndt, Mark P. Molloy, Levon M. Khachigian

From the Centre for Vascular Research (N.Y.T., V.C.M., M.M.K., F.S.S., X.L., R.P., R.G.F., L.M.K.), School of Medical Sciences, University of New South Wales, Sydney; Department of Immunology (M.C.B.), Monash University, Clayton; and Australian Proteome Analysis Facility (M.P.M.), Macquarie University, Sydney, Australia.

Correspondence to Levon M. Khachigian, PhD, DSc, Centre for Vascular Research, Department of Pathology, University of New South Wales, Sydney NSW 2052, Australia. E-mail L.Khachigian{at}unsw.edu.au

Sp1, the first identified and cloned transcription factor, regulates gene expression via multiple mechanisms including direct protein–DNA interactions, protein–protein interactions, chromatin remodeling, and maintenance of methylation-free CpG islands. Sp1 is itself regulated at different levels, for example, by glycosylation, acetylation, and phosphorylation by kinases such as the atypical protein kinase C-. Although Sp1 controls the basal and inducible regulation of many genes, the posttranslational processes regulating its function and their relevance to pathology are not well understood. Here we have used a variety of approaches to identify 3 amino acids (Thr668, Ser670, and Thr681) in the zinc finger domain of Sp1 that are modified by PKC- and have generated novel anti-peptide antibodies recognizing the PKC-–phosphorylated form of Sp1. Angiotensin II, which activates PKC- phosphorylation (at Thr410) via the angiotensin II type 1 receptor, stimulates Sp1 phosphorylation and increases Sp1 binding to the platelet-derived growth factor-D promoter. All 3 residues in Sp1 (Thr668, Ser670, and Thr681) are required for Sp1-dependent platelet-derived growth factor-D activation in response to angiotensin II. Immunohistochemical analysis revealed that phosphorylated Sp1 is expressed in smooth muscle cells of human atherosclerotic plaques and is dynamically expressed together with platelet-derived growth factor-D in smooth muscle cells of the injured rat carotid artery wall. This study provides new insights into the regulatory mechanisms controlling the PKC-–phospho-Sp1 axis and angiotensin II–inducible gene expression.

Key Words: Sp1 phosphorylation • PKC- • PDGF-D • transcription