A novel β-adrenergic response element regulates both basal and agonist-induced expression of cyclin-dependent kinase 1 gene in cardiac fibroblasts

Am J Physiol Cell Physiol. 2014 Mar 15;306(6):C540-50. doi: 10.1152/ajpcell.00206.2013. Epub 2014 Jan 29.

Abstract

Cardiac fibrosis, a known risk factor for heart disease, is typically caused by uncontrolled proliferation of fibroblasts and excessive deposition of extracellular matrix proteins in the myocardium. Cyclin-dependent kinase 1 (CDK1) is involved in the control of G2/M transit phase of the cell cycle. Here, we showed that isoproterenol (ISO)-induced cardiac fibrosis is associated with increased levels of CDK1 exclusively in fibroblasts in the adult mouse heart. Treatment of primary embryonic ventricular cell cultures with ISO (a nonselective β-adrenergic receptor agonist) increased CDK1 protein expression in fibroblasts and promoted their cell cycle activity. Quantitative PCR analysis confirmed that ISO increases CDK1 transcription in a transient manner. Further, the ISO-responsive element was mapped to the proximal -100-bp sequence of the CDK1 promoter region using various 5'-flanking sequence deletion constructs. Sequence analysis of the -100-bp CDK1 minimal promoter region revealed two putative nuclear factor-Y (NF-Y) binding elements. Overexpression of the NF-YA subunit in primary ventricular cultures significantly increased the basal activation of the -100-bp CDK1 promoter construct but not the ISO-induced transcription of the minimal promoter construct. In contrast, dominant negative NF-YA expression decreased the basal activity of the minimal promoter construct and ISO treatment fully rescued the dominant negative effects. Furthermore, site-directed mutagenesis of the distal NF-Y binding site in the -100-bp CDK1 promoter region completely abolished both basal and ISO-induced promoter activation of the CDK1 gene. Collectively, our results raise an exciting possibility that targeting CDK1 or NF-Y in the diseased heart may inhibit fibrosis and subsequently confer cardioprotection.

Keywords: CDK1; NF-Y; cardiac fibroblast proliferation; isoproterenol; β-adrenergic receptor agonist.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adrenergic beta-Agonists / pharmacology
  • Animals
  • CCAAT-Binding Factor / genetics
  • CCAAT-Binding Factor / metabolism*
  • CDC2 Protein Kinase / biosynthesis
  • CDC2 Protein Kinase / genetics
  • CDC2 Protein Kinase / metabolism*
  • Cell Proliferation
  • Cells, Cultured
  • DNA-Binding Proteins / genetics
  • Fibroblasts / metabolism
  • Fibrosis
  • Isoproterenol
  • Male
  • Mice
  • Myocardium / enzymology*
  • Myocardium / pathology*
  • Promoter Regions, Genetic / genetics
  • RNA, Messenger / biosynthesis
  • Response Elements

Substances

  • Adrenergic beta-Agonists
  • CCAAT-Binding Factor
  • DNA-Binding Proteins
  • Nfya protein, mouse
  • RNA, Messenger
  • CDC2 Protein Kinase
  • Isoproterenol