Shear stress regulates expression of death-associated protein kinase in suppressing TNFα-induced endothelial apoptosis

J Cell Physiol. 2012 Jun;227(6):2398-411. doi: 10.1002/jcp.22975.

Abstract

Death associated protein kinase (DAPK) is a positive regulator in tumor necrosis factor α (TNFα)-induced apoptotic pathway, and DAPK expression is lost in cancer cells. In the vasculature, misdirected apoptosis in endothelial cells leads to pathological conditions such as inflammation and physiological shear stress is protective against apoptosis. Using bovine aortic endothelial cells, we found that DAPK expression increased, while the auto-inhibitory phosphorylation of serine 308 decreased with shear stress at 12 dynes/cm(2) for 6 h. Quantitative RT-PCR revealed a corresponding increase in DAPK mRNA [P < 0.01]. We found that after 18-h TNFα induction, shearing cells for another 6 h significantly reduced apoptosis based on TUNEL staining [P < 0.05], although cell necrosis was not affected. Under the same conditions, we observed significantly decreased overall DAPK, as well as phospho-serine 308 DAPK [P < 0.05] compared to TNFα treatment alone. Caspase-3 and -7 activities downstream of DAPK were also attenuated. Shearing cells alone resulted in enhanced apoptosis, likely due to increased DAPK activity. Our findings were further supported by DAPK siRNA, which yielded contrary results. We present conclusive evidence for the first time that shear stress of up to 6 h up-regulates DAPK expression and activation. However, in the presence of apoptotic stimuli such as TNFα, shear stress caused decrease in DAPK activity. In fact, long-term shear stress of 24 h significantly reduced overall DAPK expression. Our findings strongly support a novel role for DAPK in mediating effects of shear stress in suppressing cytokine-activated apoptosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis Regulatory Proteins / genetics
  • Apoptosis Regulatory Proteins / metabolism*
  • Apoptosis*
  • Calcium-Calmodulin-Dependent Protein Kinases / genetics
  • Calcium-Calmodulin-Dependent Protein Kinases / metabolism*
  • Caspase 3 / metabolism
  • Caspase 7 / metabolism
  • Cattle
  • Cells, Cultured
  • Death-Associated Protein Kinases
  • Endothelial Cells / enzymology*
  • Endothelial Cells / pathology
  • Enzyme Activation
  • Flow Cytometry
  • Humans
  • In Situ Nick-End Labeling
  • Mechanotransduction, Cellular*
  • Necrosis
  • Phosphorylation
  • RNA Interference
  • RNA, Messenger / metabolism
  • Real-Time Polymerase Chain Reaction
  • Reverse Transcriptase Polymerase Chain Reaction
  • Serine
  • Stress, Mechanical
  • Time Factors
  • Transfection
  • Tumor Necrosis Factor-alpha / metabolism*
  • Up-Regulation

Substances

  • Apoptosis Regulatory Proteins
  • RNA, Messenger
  • Tumor Necrosis Factor-alpha
  • Serine
  • Death-Associated Protein Kinases
  • Calcium-Calmodulin-Dependent Protein Kinases
  • Caspase 3
  • Caspase 7