Nonmuscle Myosin-Dependent Synthesis of Type I Collagen

doi:10.1016/j.jmb.2010.06.057

Journal of Molecular Biology

Volume 401, Issue 4, 27 August 2010, Pages 564-578

https://doi.org/10.1016/j.jmb.2010.06.057 Get rights and content

Abstract

Type I collagen, synthesized in all tissues as the heterotrimer of two α1(I) polypeptides and one α2(I) polypeptide, is the most abundant protein in the human body. Here we show that intact nonmuscle myosin filaments are required for the synthesis of heterotrimeric type I collagen. Conserved 5′ stem–loop in collagen α1(I) and α2(I) mRNAs binds the RNA-binding protein LARP6. LARP6 interacts with nonmuscle myosin through its C-terminal domain and associates collagen mRNAs with the filaments. Dissociation of nonmuscle myosin filaments results in secretion of collagen α1(I) homotrimer, diminished intracellular colocalization of collagen α1(I) and α2(I) polypeptides (required for folding of the heterotrimer), and their increased intracellular degradation. Inhibition of the motor function of myosin has similar collagen-specific effects, while disruption of actin filaments has a general effect on protein secretion. Nonmuscle myosin copurifies with polysomes, and there is a subset of polysomes involved in myosin-dependent translation of collagen mRNAs. These results indicate that association of collagen mRNAs with nonmuscle myosin filaments is necessary to coordinately synthesize collagen α1(I) and α2(I) polypeptides. We postulate that LARP6/myosin-dependent mechanism regulates the synthesis of heterotrimeric type I collagen by coordinating the translation of collagen mRNAs.

Introduction

Type I collagen is the most abundant protein in the human body. It is composed of two α1(I) polypeptides and one α2(I) polypeptides, which fold into a triple helix.¹ Fibroproliferative disorders are characterized by excessive production of type I collagen by activated fibroblasts and myofibroblasts in tissues that normally do not synthesize type I collagen,2, 3, 4, 5 and they are the major causes of mortality and morbidity, being associated with 45% of deaths in the United States.⁶ There is no cure for fibrosis, and excessive collagen production is usually irreversible.⁷ All complications of fibroproliferative disorders are due to excessive collagen production, and the molecular mechanism of excessive collagen synthesis must be elucidated to develop anti-fibrotic drugs. The biosynthesis of type I collagen has multiple steps; however, recently, it became evident that regulation of the stability of collagen mRNAs and their translation constitute the predominant mechanism for high-level synthesis in multiple cell types.8, 9, 10, 11, 12

In the 5′ untranslated region of collagen α1(I), α2(I), and α1(III) mRNAs, there is a conserved 5′ stem–loop (5′SL) structure.13, 14, 15 We cloned LARP6, the protein that binds 5′SL with high affinity and specificity.¹⁶ This binding is necessary for high-level expression of type I collagen. We postulated that LARP6 binding serves to prevent premature translation of collagen mRNAs, allowing their subsequent coordinated translation on the membrane of the endoplasmic reticulum (ER).¹⁷ This coordination is evidenced by localization of collagen synthesis into discrete subcellular sites.¹⁶ Translation of collagen α1(I) and α2(I) mRNAs in close proximity to these sites may be needed to increase the local concentration of the polypeptides, which favors the formation of α1(I)/α2(I)/α1(I) heterotrimers. Heterotrimers of type I collagen are almost exclusively synthesized in all tissues,¹⁸ although the homotrimers of α1(I) polypeptides readily form if α2(I) polypeptide is not expressed.19, 20 Folding of collagen triple helix starts with disulfide bonding of two α1(I) polypeptides and one α2(I) polypeptide at the C-terminal end, with subsequent folding into a triple helix.¹ Disulfide-bonded collagen polypeptides were found to be associated with polysomes,²¹ suggesting that interchain bonding starts before the release of the polypeptides from the polysomes. Folding and posttranslational modifications of collagen polypeptides are in kinetic equilibrium, and slow folding results in hypermodification of the polypeptides. Hypermodified collagen peptides fold into an unstable triple helix, resulting in a phenotype of osteogenesis imperfecta.22, 23 Therefore, translational elongation, the rate of modification, and the rate of folding are coordinated. TRAM2 protein, as part of translocons, associates the Ca²⁺ pump Serca2b with the translocons where collagen chains are elongated. It has been proposed that this increases local Ca²⁺ concentration to stimulate collagen-specific molecular chaperones, facilitating folding of the heterotrimer.¹²

Despite cloning and characterization of LARP6, the mechanism that coordinates the synthesis of type I collagen is poorly understood. In this work, we describe one key step in the synthesis of type I collagen by profibrotic cells—the interaction of collagen mRNAs with filaments composed of nonmuscle myosin.

Section snippets

Nonmuscle myosin copurifies with 5′SL RNA

LARP6 had been cloned before as a protein that directly binds 5′SL of collagen mRNAs;¹⁶ however, other proteins that associate in complex with LARP6 and 5′SL have been unknown. To identify these proteins, we performed tobramycin affinity purification by attaching a tobramycin aptamer to the 5′SL RNA (Fig. 1a). Affinity purification using a tobramycin aptamer has been described for purification of splicing complexes.24, 25 After incubation of the collagen 5′SL/tobramycin aptamer RNA in cytosolic

Discussion

In heart fibrosis, reexpression of the fetal form of nonmuscle myosin was found only at the sites of focal fibrosis.³⁹ Mice that have the mutated 5′SL in the endogenous collagen α1(I) gene (and from which Δ5′SL MEFs used in this study were derived) develop 50% less liver fibrosis than control mice (Parsons et al., submitted). These findings suggest that the mechanism involving 5′SL and nonmuscle myosin is important for high-level collagen synthesis in vivo. Therefore, the results described here

Chemicals and cells

ML-7, blebbistatin, cytochalasin B, puromycin, and cycloheximide were purchased from Sigma. ML-7 was used at 40 μM, blebbistatin was used at 100 μM, and cytochalasin B was used at 20 μM. Cells were incubated with the drugs for 16 h before analysis. Puromycin (200 μg/ml) and cycloheximide (100 μg/ml) were added to the cells for 2 h.

Human lung fibroblasts have been described previously.¹⁶ Scleroderma fibroblasts were purchased from the European Collection of Cell Cultures (cell line BM0070) and

Acknowledgements

This work was supported by grants from the National Institutes of Health (R01DK059466), the National Scleroderma Foundation (B.S.), and the American Heart Association (L.C.).

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^†: L.C. and D.F. contributed equally to this work.

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Journal of Molecular Biology

Nonmuscle Myosin-Dependent Synthesis of Type I Collagen

Abstract

Introduction

Section snippets

Nonmuscle myosin copurifies with 5′SL RNA

Discussion

Chemicals and cells

Acknowledgements

Matrix Biol.

Clin. Chim. Acta

J. Invest. Dermatol.

Trends Cardiovasc. Med.

Chest

J. Biol. Chem.

Arch. Biochem. Biophys.

J. Biol. Chem.

Semin. Cell Dev. Biol.

J. Biol. Chem.

J. Mol. Biol.

J. Biol. Chem.

Differentiation

J. Biol. Chem.

Hepatology

J. Biol. Chem.

Anal. Biochem.

Cytokines and fibrogenesis

Semin. Liver Dis.

Reversal of hepatic fibrosis—fact or fantasy?

Hepatology

Posttranscriptional regulation of collagen alpha1(I) mRNA in hepatic stellate cells

Mol. Cell. Biol.

Differential regulation of transcription and transcript stability of pro-alpha 1(I) collagen and fibronectin in activated fibroblasts derived from patients with systemic scleroderma

Biochem. J.

Altered regulation of collagen metabolism in scleroderma fibroblasts grown within three-dimensional collagen gels

Exp. Dermatol.

TRAM2 protein interacts with endoplasmic reticulum Ca2+ pump Serca2b and is necessary for collagen type I synthesis

Mol. Cell. Biol.

Characterization of the interaction between alphaCP(2) and the 3′-untranslated region of collagen alpha1(I) mRNA

Nucleic Acids Res.

Regulatory role of the conserved stem–loop structure at the 5′ end of collagen alpha1(I) mRNA

Mol. Cell. Biol.

TRAM2 protein interacts with endoplasmic reticulum Ca²⁺ pump Serca2b and is necessary for collagen type I synthesis