Molecular mechanisms of collagen isotype-specific modulation of smooth muscle cell phenotype

Abstract

Objective: Smooth muscle cell (SMC) phenotypic modulation, an important component of atherosclerosis progression, is critically regulated by the matrix, with normal components of the healthy SMC matrix limiting modulation and atherosclerosis-associated transitional matrix proteins promoting phenotypic modulation. We sought to determine how collagen IV (which comprises the healthy artery wall) and monomeric collagen I (which comprises atherosclerotic lesions) differentially affect SMC phenotype.

Methods and results: Plating SMCs on collagen IV resulted in elevated expression of SMC contractility proteins compared to collagen I. Concurrent with enhanced contractile gene expression, collagen IV stimulates binding of SRF to CArG boxes in the promoters of smooth muscle actin and smooth muscle myosin heavy chain. Coll IV also stimulated the expression of myocardin, a critical SRF coactivator required to drive expression of SMC specific genes. In contrast to collagen IV, collagen I stimulated enhanced expression of the inflammatory protein vascular cell adhesion molecule (VCAM)-1. NF-kappaB and NFAT-binding sites in the VCAM-1 promoter are critical for collagen I-mediated expression of VCAM-1 promoter activity. However, only inhibitors of NFAT, not NF-kappaB, were able to reduce collagen I-associated VCAM expression, and collagen I but not collagen IV stimulated NFAT transcriptional activity.

Conclusions: These results show for the first time that collagen IV and collagen I differentially affect smooth muscle phenotypic modulation through multiple pathways.

Figure 1

Regulation of smooth muscle–specific gene expression by collagen isotypes. A, Rat aortic SMCs expressing luciferase constructs driven by the SMA or SM-MHC promoter were plated on Coll I or Coll IV for 6 hour. Luciferase assays were performed on cell lysates, P<0.05. B, SMCs were plated on Coll I or Coll IV for 3 days, and mRNA was extracted. SMA (B) and SM-MHC (C) expression levels were measured using quantitative RT-PCR and shown as a ratio to 18S expression, P<0.05. D, SMCs plated on Coll isotypes for 3 days were lysed and SMA protein levels were quantified using Western blotting and normalized to total protein. A representative image is shown, P<0.0001. All experiments represent 3 biological replicates, each replicate run in triplicate.

Figure 2

Coll IV promotes SRF/CArG-dependent regulation of SMA and SMMHC. SMCs were plated on either Coll I or Coll IV for 3 days in serum free media. SRF enrichment by ChIP was performed and associated DNA complexes were analyzed by quantitative RT-PCR. Primers specific to the CArG A and CArG B regions of the (A) SMA promoter (P<0.001, n=4) and the (B) SM-MHC promoter (P<0.002, n=3) were used. C, SMCs were transfected with luciferase reporters driven by the SMA promoter containing mutations in each of 3 CArG boxes. Cells were trypsinized and plated on Coll I– and Coll IV–coated plates in serum free media for 24 hours. Luciferase assays were performed. Biological replicates, n=3, in triplicate, P<0.05. D, SMCs were plated on either Coll I or Coll IV for 3 days, mRNA was extracted, and myocardin expression levels were determined using quantitative RT-PCR. Representative results are shown as a ratio of myocardin expression to 18S; n=3, in triplicate, P<0.05. E, SMCs were plated on either Coll I or Coll IV for 3 days. Cells were lysed, and SRF protein levels were determined by Western blotting. Representative blot is shown.

Figure 3

Coll I promotes VCAM-1 expression. SMC plating times on Coll I or Coll IV were varied at 24 hours, 48 hours, or 5 days, and VCAM-1 (A) mRNA expression (n=3, in triplicate, P<0.001) and (B) protein expression (n=3, in triplicate, P<0.001) were determined. A representative image is shown.

Figure 4

The NFAT/NF-κB binding site in the VCAM-1 promoter is required for Coll I–induced VCAM-1 expression. SMCs expressing luciferase constructs driven by the VCAM-1 promoter or a mutant VCAM-1 promoter lacking the NF-κB and NFAT cis elements were plated on Coll I or Coll IV for 6 or 24 hours. Luciferase assays were then performed on cell lysates. n=3, in triplicate, P<0.05.

Figure 5

Coll I promotes NFAT-dependent VCAM-1 expression. A, SMCs were pretreated with SN50 (30 μg/mL), CsA (10 μmol/L), or a combination of the two for 1 hour. Cells were plated onto Coll I–coated dishes in the presence of the inhibitors for 24 hours. VCAM-1 protein expression was determined. A representative image is shown. n=3, in triplicate, P<0.01. B, SMCs were pretreated with CsA for 1 hour and plated onto Coll I–coated dishes in the presence of the inhibitors. After 24 hours, mRNA was extracted and VCAM-1 expression was determined by quantitative RT-PCR (representative results from a triplicate experiment; n=3, in triplicate, P<0.05) C, SMCs were transfected with VCAM-1 luciferase constructs. Cells were trypsinized and plated onto Coll I– and Coll IV–coated plates in the presence of CsA or A-285222 (10 μmol/L). Luciferase assays were performed after 6 hours of plating. Biological replicates in triplicate; n=3, in triplicate, P<0.05. D, SMCs expressing an NFATc-driven luciferase construct were trypsinized and plated Coll I– and Coll IV–coated plates in the presence of CsA for 6 and 24 hours. Luciferase assays were performed. n=3, in triplicate; *different from Coll IV, Coll IV+CsA, and Coll I+CsA, P<0.05.

Figure 6

Molecular mechanisms of matrix-dependent SMC phenotypic modulation. Proteins colored in green are proposed to favor Coll IV–dependent activation of SMC differentiation marker expression (Myoc, SMMHC, SMA) and the contractile phenotype; dashed lines indicate that the signaling pathways to this molecular fingerprint are unknown. Proteins colored in red are proposed to favor Coll I–dependent activation of the inflammatory phenotype and VCAM-1 via calcineurin (CaN) and NFAT. NF-κB is depicted as a dashed transcription factor because although it was shown herein that Coll I does not mediate NF-κB–dependent regulation of VCAM-1, NF-κB has been shown to regulate VCAM-1 in response to other stimuli such at IL-1β. It is currently unknown which integrins/receptors mediate matrix-dependent regulation of SMC phenotype (gray receptors with ?). CsA indicates cyclosporine A; Ac, acetylation.