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. 2008 May 23;283(21):14542-51.
doi: 10.1074/jbc.M802139200. Epub 2008 Mar 24.

Monocyte chemotactic protein (MCP)-1 promotes angiogenesis via a novel transcription factor, MCP-1-induced protein (MCPIP)

Jianli Niu  1 , Asim Azfer, Olga Zhelyabovska, Sumbul Fatma, Pappachan E Kolattukudy
Affiliations

Monocyte chemotactic protein (MCP)-1 promotes angiogenesis via a novel transcription factor, MCP-1-induced protein (MCPIP)

Jianli Niu et al. J Biol Chem. .

Abstract

Monocyte chemotactic protein-1 (MCP-1) has been recognized as an angiogenic chemokine. The molecular mechanism of MCP-1-mediated angiogenesis remains unknown. We recently identified a novel transcription factor, designated MCP-1-induced protein (MCPIP), in human monocytes after treatment with MCP-1. We investigated whether MCP-1-induced angiogenesis is mediated via MCPIP. Treatment of human umbilical vein endothelial cells (HUVECs) with MCP-1 induced expression of MCPIP and capillary-like tube formation. Knockdown of MCPIP by small interfering RNA (siRNA) suppressed MCP-1-induced angiogenesis-related gene VEGF and HIF-1alpha expression as well as tube formation. Transfection of HUVECs with an MCPIP expression vector induced angiogenesis-related genes and tube formation. Chromatin immunoprecipitation analysis revealed that cadherin (cdh) 12 and cdh19 are in vivo targets of MCPIP. Transfection of HUVECs with MCPIP expression vector activated the expression of cdh12 and cdh19 genes. Knockdown of cdh12 or cdh19 expression markedly inhibited MCPIP-induced capillary-like tube formation. Moreover, knockdown of MCPIP also significantly suppressed MCP-1-induced cdh12 and cdh19 gene expression. Our data strongly suggest that MCP-1-induced angiogenesis is mediated via MCPIP, at least in part through transcriptional activation of cdh12 and cdh19.

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Figures

FIGURE 1.
FIGURE 1.
MCP-1-induced angiogenesis is mediated via induction of MCPIP. HUVECs were treated with MCP-1 with or without transfection with MCPIP-specific or nonspecific siRNA for 24 h. Expression of MCPIP was detected by RT-PCR (A), real-time PCR (B), and immunoblot (C) analyses. β-Actin was amplified as an internal control for RT-PCR. D, histogram depicting the average MCPIP expression levels in the examined groups as assessed by immunoblot analysis. E, phase-contrast photomicrographs of HUVECs treated with MCP-1 with or without MCPIP-specific or nonspecific siRNA for 24 h (original magnification ×100), and the quantitative tube formation assay (F). *, p < 0.001 for treatment with MCP-1 and with nonspecific siRNA.
FIGURE 2.
FIGURE 2.
Real-time PCR analysis of HIF-α (A) and VEGF (B) expression in HUVECs treated with MCP-1 with or without MCPIP-specific or nonspecific siRNA. *, p < 0.05 for treatment with MCP-1 and with nonspecific siRNA. #, p < 0.05 versus GFP vector-transfected HUVECs.
FIGURE 3.
FIGURE 3.
Expression of MCPIP induces capillary-like tube formation in HUVECs. HUVECs were transfected with the MCPIP-GFP expression vector or GFP control for 24 h, and expression of MCPIP was detected by real-time PCR (A) and immunoblot (B) analyses. *, p < 0.001 versus GFP vector-transfected HUVECs. C, phase-contrast photomicrographs (original magnification ×100) of HUVECs seeded on the surface of the polymerized fibrin gels for 24 h after transfection with MCPIP-GFP expression vector or GFP control. D, mean number of tube branch points in randomly selected 5 high power fields (×40) of views was quantified. *, p < 0.05 versus GFP vector-transfected HUVECs.
FIGURE 4.
FIGURE 4.
Expression of MCPIP induces angiogenesis-related properties in HUVECs. A, confluent HUVECs monolayers were wounded by scraping and transfected with the expression vector for MCPIP-GFP or GFP control in serum-free medium. Cell migration to the wound surface was monitored from 0 to 24 h and quantitated at 24 h (B). Proliferation was detected by BrdU incorporation (C), and apoptosis was determined by TUNEL staining (D and E) in HUVECs transfected with expression vector for MCPIP-GFP or GFP control for 24 h. *, p < 0.05 versus GFP-vector transfected HUVECs.
FIGURE 5.
FIGURE 5.
Induction of cdh12 and cdh19 by MCPIP and specific binding of MCPIP to cdh12 and cdh19. A, HEK293 cells were transfected with the MCPIP-GFP expression vector or GFP control for 12 h. RNA was isolated and subjected to RT-PCR using primers for cdh12 and cdh19 sequences. β-Actin was amplified as an internal control. B and C, gel retardation assays were performed as described under “Experimental Procedures” with a 32P-labeled 415-bp cdh12 gene fragment and a 207-bp cdh19 fragment with or without the indicated excess of unlabeled specific gene fragment or nonspecific DNA.
FIGURE 6.
FIGURE 6.
MCPIP induces cdh12 and cdh19 expression in HUVECs. A and B, HUVECs were transfected with MCPIP-GFP expression vector or GFP control for 24 h, and expression of cdh12 and cdh19 was detected by real-time PCR. The expression of both cdh12 and cdh19 at protein levels was demonstrated by immunoblot analysis (C and D). VE-cadherin, an endothelial cell-specific cadherin required for angiogenesis, was also found to be induced by MCPIP at both transcript and protein levels, assayed by real-time PCR (E) and Western blot (F). *, p < 0.05 versus GFP vector-transfected HUVECs.
FIGURE 7.
FIGURE 7.
Contribution of cdh12 and cdh19 expression to MCPIP-mediated angiogenesis. A and B, real-time PCR analysis of cdh12 and cdh19 mRNA in HUVECs transfected with the expression vector for MCPIP-GFP with or without cdh12- and cdh19-specific or nonspecific siRNA showed that only siRNA specific for the particular cdh gene showed knockdown. *, p < 0.05 versus MCPIP vector-transfected HUVECs. C and D, histograms depicting the average cdh12 and cdh19 expression levels in the examined groups shown in the Western blot. Lane 1, GFP; lane 2, MCPIP; lane 3, MCPIP+cdh12 siRNA; lane 4, MCPIP+cdh19 siRNA; lane 5, MCPIP+cdh12,19 siRNA. *, p < 0.05; #, p < 0.01 versus MCPIP vector-transfected HUVECs. E, phase-contrast photomicrographs (original magnification ×100) of HUVECs seeded on the surface of the polymerized fibrin gels for 24 h after transfection with MCPIP expression vector, MCPIP+cdh12 siRNA, MCPIP+cdh19 siRNA, or MCPIP+cdh12, 19 siRNA. F, mean number of tube branch points in randomly selected 5 high-power field (×100) of views were quantified and expressed as a percentage of MCPIP-treated group; *, p < 0.05 versus MCPIP vector-transfected HUVECs.
FIGURE 8.
FIGURE 8.
MCP-1 treatment induces expression of cdh12 and cdh19 in HUVECs and siRNA specific for MCPIP inhibits their expression. A and B, HUVECs were treated with MCP-1 (100 ng/ml), in the presence or absence of MCPIP-specific or nonspecific siRNA for 24 h, and mRNA expression of cdh12 and cdh19 was assessed by real time-PCR demonstrating that knockdown of MCPIP inhibited MCP-1 induced expression of cdh12 and cdh19.*, p < 0.05 versus MCP-1- or nonspecific siRNA-treated HUVECs. β-Actin was amplified as an internal control. C and D, histograms depicting the average cdh12 and cdh19 expression levels in the examined groups shown in the Western blot. Lane 1, control; lane 2, MCP-1; lane 3, MCP-1+nonspecific siRNA; lane 4, MCP-1+MCPIP siRNA. *, p < 0.05 versus MCP-1-treated HUVECs or MCP-1 with nonspecific siRNA-treated HUVECs.
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