Mechanisms in cardiac fibroblast growth: an obligate role for Skp2 and FOXO3a in ERK1/2 MAPK-dependent regulation of p27kip1.
Cardiac fibroblast hyperplasia associated with enhanced matrix deposition is a major determinant of tissue remodeling in several disease states of the heart. However, mechanisms controlling cell cycle progression in cardiac fibroblasts remain unexplored. Identification of cell cycle regulatory elements in these cells is important to develop strategies to check adverse cardiac remodeling under pathological conditions. This study sought to probe the mechanisms underlying ERK1/2-mediated p27(Kip1) regulation in mitogenically stimulated cardiac fibroblasts. Addition of 10% fetal calf serum to quiescent cultures of adult rat cardiac fibroblasts promoted ERK1/2 activation, as evidenced by its phosphorylation status. Reduction in [(3)H]thymidine incorporation into DNA increased population doubling time, flow cytometry, and Western blot analysis showing reduced levels of cyclins D and A, p27(Kip1) induction, and retinoblastoma protein (Rb) hypophosphorylation in ERK1/2-inhibited cells indicated ERK1/2 dependence of G1-S transition in cardiac fibroblasts. Lack of p27(Kip1) protein in serum-stimulated, ERK1/2-active cells was associated with increased levels of Skp2, an E3 ubiquitin ligase for p27(Kip1), whose knockdown by RNA interference induced p27(Kip1) expression. Further, forced expression of Skp2 in ERK1/2-inhibited cells downregulated p27(Kip1). Transcriptional upregulation of p27(Kip1) mRNA in ERK1/2-inhibited cells, demonstrated by real-time PCR, correlated with forkhead box O 3a (FOXO3a) transcription factor activation, shown by gel shift assay. FOXO3a knockdown attenuated p27(Kip1) mRNA and protein expression in ERK1/2-inhibited cells. We provide evidence for the first time that, in cardiac fibroblasts, activated ERK1/2 regulates p27(Kip1) expression transcriptionally and posttranslationally via FOXO3a- and Skp2-dependent mechanisms. Additionally, this study uncovers interesting interactions between critical cell cycle regulatory elements that are only beginning to be understood.
Division of Cellular and Molecular Cardiology, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Trivandrum, India.
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S
Pramod
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K
Shivakumar
D000818:Animals; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000502:physiology*; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors / Q000502:physiology*; D020935:MAP Kinase Signaling System / Q000502:physiology*; D008297:Male; D008934:Mitogens / Q000494:pharmacology; D023421:Models, Animal; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D044786:S-Phase Kinase-Associated Proteins / Q000502:physiology*
C499515:FOXO3 protein, rat; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors; D008934:Mitogens; D044786:S-Phase Kinase-Associated Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27
ERK1/2; FOXO3a; Skp2; cardiac fibroblasts; p27