Monocyte chemoattractant protein-1 induces a novel transcription factor that causes cardiac myocyte apoptosis and ventricular dysfunction

Abstract

Monocyte chemoattractant protein-1 (MCP-1; CCL2)-mediated inflammation plays a critical role in the development of ischemic heart disease (IHD). However, the gene expression changes caused by signal transduction, triggered by MCP-1 binding to its receptor CCR2, and their possible role in the development of IHD are not understood. We present evidence that MCP-1 binding to CCR2 induces a novel transcription factor (MCP-induced protein [MCPIP]) that causes cell death. Gene microarray analysis showed that when expressed in hiuman embryonic kidney 293 cells, MCPIP induced apoptotic gene families before causing cell death. Mutagenesis studies showed that the structural features required for transcription factor-like activity were also required for causing cell death. Activation of caspase-3 was detected after MCPIP transfection and Z-VAD-fmk partially inhibited cell death. Cardiomyocyte-targeted expression of MCP-1 in mice caused death by heart failure at 6 months of age. MCPIP expression increased in parallel with the development of ventricular dysfunction. In situ hybridization showed the presence of MCPIP transcripts in the cardiomyocytes and immunohistochemistry showed that MCPIP was associated with the cardiomyocyte nuclei of apoptotic cardiomyocytes. CCR2 expression in cardiomyocytes increased with the development of IHD. MCPIP production induced by MCP-1 binding to CCR2 in the cardiomyocytes is probably involved in the development of IHD in this murine model. MCPIP transcript levels were much higher in the explanted human hearts with IHD than with nonischemic heart disease. These results provide a molecular insight into how chronic inflammation and exposure to MCP-1 contributes to heart failure and suggest that MCPIP could be a potential target for therapeutic intervention.

Figure 1

A, Schematic representation of MCPIP showing putative domain structure of the human MCPIP protein. B, Induction of human MCPIP gene expression in human monocytes by treatment with 7 nmol/L MCP-1 as detected by RNA blot. C, Effect of anti-CCR2 antibodies on MCP-1 induction of MCPIP. MCPIP transcript was measured by RT-PCR in RAW-264.7 cells treated with 20 μg MCP-1 in the presence of the indicated amounts of mouse monoclonal anti-CCR2 (GeneTex).

Figure 2

A, Confocal microscope images of HEK293 cells expressing human MCPIP-GFP (top) and GFP control (bottom). GFP fluorescence from expressed protein (green); propodium iodide (PI)–stained cell nuclei (red); merged green and red fluorescence (yellow). B, Cell death by transfection with hMCPIP-GFP of HEK293 cells () and GFP alone (▪). Cells were transfected with MCPIP–GFP or GFP alone, harvested at either day 1 or day 5 after transfection, >200 cells were examined for each time point, and experiments were repeated four times. Cell death was detected by TUNEL and trypan blue. C, Immunoblot analysis of PARP cleavage in HEK293 cells transfected with MCPIP–GFP and GFP alone using PARP antibodies from Abcam. D, Immunoblot analysis of caspase-3 species recovered with avidin after labeling the active caspases with biotin-Z-VAD-fmk showing caspase-3 activation. The inhibitor treatment was done 3 days after transfection, and the blots were quantified by densitometry. E, Inhibition of HEK293 death induced by MCPIP by Z-VAD-fmk. *P<0.05

Figure 3

In vitro transactivation of luciferase reporter gene by cotransfected hMCPIP or its mutants in cell cultures (A) and cell death induced by hMCPIP or its mutants (B). Luciferase activity resulting from expression of pCGAL vector alone (vector), MCPIP–pCGAL fusion protein, or fusion proteins with mutants of MCPIP in HEK293 cell was measured. Cell death was assessed by trypan blue staining. PRO1 and PRO2 indicate proline-rich domain mutants 1 and 2; ZNF, zinc finger mutant.

Figure 4

Age-dependent increase in MCPIP (A) and MCP-1 (B) gene expression in the hearts of wild-type (▪) and MCP-1 mice () and decrease in fractional shortening (C). Gene expression was measured by quantitative real-time RT-PCR.

Figure 5

A, In situ hydridization showing elevated expression of MCPIP in the cardiomyocytes of MCP mice. B, Immunohistochemical detection of MCPIP in the hearts of MCP mice. Condensed nuclei staining (brown) with MCPIP was observed in cardiomyocytes and infiltrating inflammatory cells in MCP mice of 2 and 4 months of age (IV and V); a strong staining for MCPIP was more prominent in the cardiomyocytes showing vacuolization (VI; arrows) in 6-month-old MCP mice with heart failure. MCPIP staining was also found in vascular endothelial and smooth muscle cells (data not shown). The hearts from age-, sex-matched wild-type controls showed a weak nuclear staining (original magnification × 400). C, Double staining on the heart tissue showed that most cells with high expression of MCPIP (detected by immunostaining) also were apoptotic as indicated by TUNEL.

Figure 6

Quantitative real time PCR measurement of expression of CCR2 in the myocardium of MCP () and wild-type mice (▪; A) and in situ hybridization showing expression of CCR2 in cardiomyocytes in 6-month-old of MCP-1 mice (B). *P<0.05 vs wild-type (n = 6 each group).