Contribution of Mer receptor tyrosine kinase and the intracellular signaling pathway in apoptotic cell-induced HGF production

Abstract

대식세포 (macrophage)의한 사멸화 세포 (apoptotic cells)의 탐식은 간세포 성장인자 (Hepatocyte growth factor; HGF) 분비를 유도한다. Mer 수용기 타이로신 키나아제 (Mer receptor tyrosine kinase; Mer)는 중간의 다리 역할을 하는 분자인 Gas6 (growth arrest specific 6)를 통해 사멸화 세포 인지와 포식 과정에 이용되어져 왔다. 현 연구에서, 사멸화 세포에 대한 반응으로 HGF 유도에 있어 Mer의 기여와 세포내 신호전달 경로가 연구되어졌다. HGF mRNA (messenger RNA; 메신저 RNA)와 단백질 발현을 유도하는 조절기전은 사멸화 세포를 대식세포에 노출시킴으로서 검증되어졌다. 사멸화 된 Jurkat 세포와 RAW 264.7 대식세포와의 상호작용은 HGF mRNA와 단백질 발현의 증가를 나타냈다. 사멸화 세포를 RAW 264.7 대식세포에 노출시 RhoA, PI3K/Akt 그리고 p38 MAPK, ERK, JNK를 포함한 MAP 키나아제들의 활성이 증가되었다. 약물학적 억제제들이나 RhoA siRNA (small interfering RNA; 짧은 간섭 RNA)에 의한 RhoA/Rho 키나아제 경로의 하향-조절은 사멸화 세포에 대한 반응으로서 Akt와 MAP 키나아제들의 인산화를 통한 HGF mRNA와 단백질 발현을 억제하였다. PI3K의 억제는 Akt와 MAP 키나아제들의 인산화를 억제하였다. p38 MAPK, ERK와 달리 JNK 억제는 Akt의 인산화를 억제하였다. PI3K와 MAP 키나아제들의 약물학적 억제제는 HGF mRNA와 단백질 발현을 역시 억제하였다.;Clearance of apoptotic cells by macrophages induces hepatocyte growth factor (HGF) secretion. Mer receptor tyrosine kinase (Mer) has been implicated in the process of apoptotic cell recognition and engulfment through growth arrest-specific gene 6 (Gas6), an intermediate-bridging molecule. In the present study, the regulatory mechanisms of HGF mRNA and protein expression were examined in macrophages upon exposure to apoptotic cells. The contribution of Mer and the intracellular signaling pathway in HGF induction was also investigated in response to apoptotic cells. The interaction of RAW 264.7 macrophages with apoptotic Jurkat cells, but not with viable cells, resulted in the expression of HGF mRNA and proteins. Exposure of RAW 264.7 cells to apoptotic cells induced activation of RhoA, PI3K/Akt, and MAP kinases, including p38 MAPK, ERK, and JNK. Down-regulation of the RhoA/Rho kinase pathway by pharmacological inhibitors or a RhoA-specific siRNA suppressed HGF mRNA and protein expression by macrophages in response to apoptotic cells through the phosphorylation of Akt and the MAP kinases. Inhibition of PI3K decreased phosphorylation of Akt and the MAP kinases. Inhibition of JNK, but not p38 MAPK and ERK, reduced Akt phosphorylation. The pharmacological inhibitor of PI3K and the MAP kinases blocked HGF mRNA and protein expression. A ligand of Mer, Gas6 increased HGF mRNA and protein expression with the similar time course with apoptotic stimulation. Gas6 activated RhoA/Rho kinase/PI3K/MAP kinases signaling pathway is required for expression of Gas6 HGF mRNA and protein. A specific Mer-neutralizing antibody, Mer siRNA, or a recombinant Mer extracellular domain fused to the Fc domain of human IgG (Mer/Fc) inhibited apoptotic cell-induced HGF mRNA, protein expression, RhoA activity, and the phosphorylation of Akt and MAP kinases. A specific Mer-neutralizing antibody or Mer siRNA also inhibited Gas6-induced activation of RhoA and HGF expression at mRNA and protein levels. In contrast, apoptotic cell-induced TGF-β1 mRNA expression was not inhibited by a specific Mer-neutralizing antibody or Mer/Fc. Overall, the data provide evidence that activation of the RhoA/Rho kinase/PI3K/MAP kinases signaling pathway up-regulates transcriptional HGF production in response to apoptotic cells or Gas6. Mer activation mediates the effect of apoptotic cells on HGF production through this RhoA-dependent pathway. Mer and the post-receptor signaling pathway may be a novel target for enhancing the potential effects of apoptotic cells on epithelial regeneration and tissue replacement after tissue injury.