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. 2009 Jun 26;284(26):17564-74.
doi: 10.1074/jbc.M109.013987. Epub 2009 Apr 30.

Transforming growth factor-beta promotes recruitment of bone marrow cells and bone marrow-derived mesenchymal stem cells through stimulation of MCP-1 production in vascular smooth muscle cells

Fan Zhang  1 , Shirling Tsai, Kaori Kato, Dai Yamanouchi, Chunjie Wang, Shahin Rafii, Bo Liu, K Craig Kent
Affiliations

Transforming growth factor-beta promotes recruitment of bone marrow cells and bone marrow-derived mesenchymal stem cells through stimulation of MCP-1 production in vascular smooth muscle cells

Fan Zhang et al. J Biol Chem. .

Abstract

Bone marrow-derived progenitor cells have recently been shown to be involved in the development of intimal hyperplasia after vascular injury. Transforming growth factor-beta (TGF-beta) has profound stimulatory effects on intimal hyperplasia, but it is unknown whether these effects involve progenitor cell recruitment. In this study we found that although TGF-beta had no direct effect on progenitor cell recruitment, conditioned media derived from vascular smooth muscle cells (VSMC) stimulated with TGF-beta induced migration of both total bone marrow (BM) cells and BM-mesenchymal stem cells (MSC) and also induced MSC differentiation into smooth muscle like cells. Furthermore, overexpression of the signaling molecule Smad3 in VSMC via adenovirus-mediated gene transfer (AdSmad3) enhanced the TGF-beta's chemotactic effect. Microarray analysis of VSMC stimulated by TGF-beta/AdSmad3 revealed monocyte chemoattractant protein-1 (MCP-1) as a likely factor responsible for progenitor cell recruitment. We then demonstrated that TGF-beta through Smad3 phosphorylation induced a robust expression of MCP-1 in VSMC. Recombinant MCP-1 mimicked the stimulatory effect of conditioned media on BM and MSC migration. In the rat carotid injury model, Smad3 overexpression significantly increased MCP-1 expression after vascular injury, consistent with our in vitro results. Interestingly, TGF-beta/Smad3-induced MCP-1 was completely blocked by both Ro-32-0432 and rotterlin, suggesting protein kinase C-delta (PKCdelta) may play a role in TGF-beta/Smad3-induced MCP-1 expression. In summary, our data demonstrate that TGF-beta, through Smad3 and PKCdelta, stimulates VSMC production of MCP-1, which is a chemoattractant for bone marrow-derived cells, specifically MSC. Manipulation of this signaling system may provide a novel approach to inhibition of intimal hyperplasia.

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Figures

FIGURE 1.
FIGURE 1.
TGF-β stimulates SMC to express factors that attract bone marrow progenitor cells. Results are for rat bone marrow cell chemotaxis assays. Recombinant TGF-β (5 ng/ml) alone had no effect on total bone marrow cell migration as compared with positive control SDF-1α (100 ng/ml) (A) or on isolated rat MSC migration (B). However, conditioned media from VSMC stimulated with TGF-β was found to induce both total bone marrow cell migration (C) and isolated rat MSC migration (D) (n = 3). *, p < 0.05 compared with control.
FIGURE 2.
FIGURE 2.
Smad3 overexpression enhances the stimulatory effect of TGF-β on SMC. VSMC were infected with control virus (AdGFP) or AdSmad3 and stimulated with TGF-β. Conditioned media was then used in chemotaxis assays. Treatment with AdSmad3 in addition to TGF-β further induced VSMC secretion of factors that attract both total bone marrow cells (A) and isolated MSC (B) (n = 3). *, p < 0.05, compared with control; #, p < 0.05 compared with TGF-β.
FIGURE 3.
FIGURE 3.
TGF-β stimulates BMPC migration through Smad3 signaling. A, Western blot showing inhibition of phosphorylation of both endogenous and adenovirus-derived Smad3 by SB431542 (10 μg/ml). Shown is a chemotaxis assay using whole bone marrow cells (B) or isolated rat MSC (C) and conditioned media from VSMC pretreated with SB431542 and TGF-β/AdSmad3. Data are expressed as -fold change compared with conditioned media from VSMC infected with AdGFP only. D, Western blot showing down-regulation of total Smad3 in cells treated with Smad3 siRNA. Chemotaxis assay using total bone marrow (E) and isolated rat MSC (F) and conditioned media from VSMC transfected with Smad3 siRNA and treated with TGF-β. Data expressed as -fold change compared with conditioned media from VSMC transfected only with Lipofectamine; n = 3; *, p < 0.05.
FIGURE 4.
FIGURE 4.
TGF-β, through Smad3 signaling, induces VSMC production of MCP-1. A, TGF-β induced VSMC production of MCP-1 in a dose-dependent manner, as shown by ELISA. B, enhancement of Smad3 signaling by infection with AdSmad3 also induced MCP-1 production and enhanced the stimulatory effect of TGF-β, as shown by both ELISA and RT-PCR (C). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. D, blockade of Smad3 phosphorylation using SB431542 (10 μg/ml) inhibited TGF-β induced MCP-1 production as measured by ELISA. E, knockdown of Smad3 using a Smad3 siRNA also inhibited TGF-β induced MCP-1. (n = 4; *, p < 0.05 compared with control).
FIGURE 5.
FIGURE 5.
MCP-1 mediates TGF-β induced bone marrow and progenitor cell migration. Recombinant MCP-1 (20 ng/ml) induced both total bone marrow cell (A) and rat MSC migration (B) in chemotaxis assays. MCP-1 gene silencing using a MCP-1 siRNA inhibited TGF-β/Smad3-induced MCP-1 production both on MCP-1 ELISA (C) and RT-PCR (D). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Conditioned media from VSMC treated first with MCP-1 siRNA, then AdSmad3 and TGF-β attracted fewer total bone marrow cells (E) and rat MSC (F) when compared with conditioned media from cells treated with control siRNA or no siRNA. The addition of an MCP-1 blocking antibody (Ab) to conditioned media from VSMC treated with AdSmad3 and TGF-β also attracted fewer total bone marrow cells (G) and rat MSC (H) when compared with conditioned media from VSMC treated with only AdSmad3 and TGF-β. n = 3; *, p < 0.05 compared with TGF-β or TGF-β/AdSmad3.
FIGURE 6.
FIGURE 6.
Conditioned media from VSMC stimulated with TGF-β induces MSC differentiation into SMC. MSC were cultured in conditioned media from RSMC (A), VSMC treated with TGF-β (B), or VSMC treated with TGF-β/AdSmad3 (C) for 3 days. (D) MSC were treated with conditioned media from RSMC for 3 days. Protein lysates were analyzed by Western blotting using antibodies against α-SMA, calponin, and desmin. Tubulin served as a loading control. E, MSC were treated with or without TGF-β (5 μg/ml) for 3 days, and protein lysates were analyzed by Western blotting using antibodies against α-SMA, calponin, and β-tubulin.
FIGURE 7.
FIGURE 7.
Up-regulation of Smad3 in vivo increases MCP-1 expression and injury of arteries induce migration of BM in the arterial wall. A and B, MCP-1 IHC in rat carotid arteries 5 days after balloon injury and infection with AdNull or AdSmad3. C, semiquantitative immunohistochemistry showing increased MCP-1 expression in arteries infected with AdSmad3. *, p < 0.05. D and E, after implantation of PKH26-labeled BM in rat circulation for 24 h, PKH26-labeled BM (white arrowheads) are observed in uninjured arteries (C) but not in injured arteries (D).
FIGURE 8.
FIGURE 8.
Inhibition of PKC prevents TGF-β/Smad3-induced MCP-1 expression by RSMC. A, MCP-1 ELISA showing inhibition of TGF-β/Smad3-induced MCP-1 after pretreatment with the PKC inhibitor Ro-32-0432 (5 μm). B, the effect of Ro-32-0432 was dose-dependent (concentration in μm). C, MCP-1 ELISA showing inhibition of TGF-β/Smad3-induced MCP-1 by the PKCδ-specific inhibitor rotterlin (concentration in μm). D, conversely, overexpression of PKCδ alone induces MCP-1 expression in RSMC. Chemotaxis assays using conditioned media from VSMC pretreated with rotterlin (2 μm) then stimulated with TGF-β/AdSmad3 showed that inhibition of PKCδ decreased the chemoattractive properties of conditioned media for both total bone marrow cells (E) and rat MSC (F) (n = 3). *, p < 0.05.
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