Elsevier

Experimental Cell Research

Volume 282, Issue 2, 15 January 2003, Pages 90-100
Experimental Cell Research

Regular article
TGF-β1-mediated fibroblast–myofibroblast terminal differentiation—the role of smad proteins

https://doi.org/10.1016/S0014-4827(02)00015-0Get rights and content

Abstract

It is now clear that resident myofibroblasts play a central role in the mediation of tissue fibrosis. The aim of the work outlined in this study is to increase our understanding of the mechanisms which drive the phenotypic and functional changes associated with the differentiation process. We have used an in vitro model of transforming growth factor-β1 (TGF-β1)-induced pulmonary fibroblast–myofibroblast differentiation to examine the role of the TGF-β1 Smad protein signaling intermediates, in alterations of fibroblast phenotype and function associated with terminal differentiation. TGF-β1 induced marked alteration in cell phenotype, such that cells resembled “epithelioid-postmitotic fibroblasts.” This was associated with marked reorganization of the actin cytoskeleton and upregulation of αSMA gene expression. TGF-β1 stimulation also induced αSMA protein expression with increased incorporation of αSMA into stress fibers. Following stimulation with TGF-β1, subsequent addition of serum-free medium did not reverse TGF-β1-induced morphological change, suggesting that TGF-β1 induced a relatively stable alteration in fibroblast cell phenotype. Functionally, these phenotypic changes were associated with induction of type I, type III, and type IV collagen gene expression and an increase in the concentrations of the respective collagens in the cell culture supernatant. The role of Smad proteins in terminal differentiation of fibroblasts was examined by transfection of cells, with expression vectors for the TGFβ1 receptor-regulated Smads (R-Smads) or the co-Smad, Smad 4. Transfection with Smad2 but not Smad3 resulted in TGF-β1 independent alteration in fibroblast cell phenotype, up-regulation of αSMA mRNA and reorganization of the actin cytoskeleton. Transfection with Smad4 also induced alteration in cell phenotype, although this was not as pronounced as the effect of overexpression of Smad2. Overexpression of the Smad2, Smad3, or Smad4 proteins was associated with increased production of all collagen types. The study suggests that the phenotypic and functional changes associated with TGF-β1-induced fibroblast terminal differentiation are differentially regulated by Smad proteins.

Introduction

Increased activity and proliferation of resident fibroblasts is central to fibrosis in all tissues. The fibroblast is the most abundant cell type in normal connective tissues and plays a central role in the synthesis, degradation, and remodeling of the extracellular matrix in both health and disease. Though fibroblastic cells are traditionally considered to have a relatively uniform morphology, they are endowed with multiple functional properties and several cytoskeletal differentiation markers have been described, which suggests that a phenotypic heterogeneity, related to distinct biological functions, also exists [1]. Fibroblasts normally express only two actin isoforms (β and γ). Recent studies, however, have demonstrated that in areas of fibrosis, a subgroup of fibroblasts exists which express the smooth muscle isoform of α-actin (αSMA) that is normally expressed constitutively only in smooth muscle cells. It is now clear that these cells represent a subpopulation of specialized fibroblasts that have developed a contractile phenotype which is expressed in a number of pathological settings associated with wound healing and fibrosis [1].

The cytokine transforming growth factor β1 (TGF-β1) has been widely recognized as a key mediator of wound healing. Its aberrant expression has also been implicated in a number of fibrotic and inflammatory lesions [2]. Recent studies have demonstrated that in addition to its direct effect on extracellular matrix turnover, it may stimulate fibroblast–myofibroblast differentiation, as it is capable of up-regulating αSMA in fibroblasts both in vitro and in vivo [3], [4], [5].

The aim of the current study was to examine the functional consequences of TGF-β1-mediated activation of αSMA in fibroblasts and their differentiation into “myofibroblasts.” Specifically, human lung fibroblasts stimulated with TGF-β1 were used as a model of fibroblast–myofibroblast differentiation. Consequences of differentiation on αSMA synthesis and distribution were subsequently related to alterations in cell phenotype and functional alterations assessed by changes in collagen turnover. Finally, we examined the role of the intracellular mediators of TGF-β1 signaling, the Smad proteins, in TGF-β1-mediated alterations in fibroblast phenotype and function.

Section snippets

Cell culture

Human lung fibroblasts (AG02262) were purchased from the N.I.A. Ageing Cell Repository Corriel Institute (Camden, NJ). Cells were cultured in Dulbecco’s modified Eagle’s medium/HAM’s F-12 and supplemented with 2 mM l-glutamine, 100 U/ml of penicillin/streptomycin, 0.5 mg/ml of insulin, 0.5 mg/ml of transferrin, and 10% fetal calf serum (FCS).

The effect of TGF-β1 stimulation was determined by the addition of recombinant TGF-β1 to confluent growth-arrested monolayers of cells, as previously

Morphology

In culture human lung fibroblasts (AG02262) constitutively expressed vimentin and αSMA (Fig. 1). Under all experimental conditions, cells were consistently negative for cytokeratin (data not shown). Under resting, serum-free unstimulated conditions cells displayed a typical small spindle-shaped appearance (Fig. 1). Following stimulation with recombinant TGF-β1 (10 ng/ml) cells became larger and polygonal (Fig. 1). These changes were apparent 48 h after the addition of TGF-β1 and continued to

Discussion

The aim of the work outlined in the current paper was to characterize the structural and functional consequences of differentiation of human fibroblasts into myofibroblasts and to identify the mechanism by which these changes may be mediated by TGF-β1. Myofibroblasts are thought to be terminally differentiated cells with morphological features intermediate between those of fibroblasts and smooth muscle cells. Thus, the cells retain the biological properties of fibroblasts synthesizing

Acknowledgements

R.A.E. is supported by the National Kidney Research Fund, and Ya Chung Tian is supported by a Research Fellowship from Chang Gung Memorial Hospital, Taipei, Taiwan. A.O.P. is in receipt of a GlaxoSmithKline Senior Fellowship.

References (30)

  • A.O. Phillips et al.

    Elevated D-glucose concentrations modulate TGF-β1 synthesis by human cultured renal proximal tubular cellsThe permissive role of platelet derived growth factor

    Am. J. Pathol.

    (1995)
  • D.S. Romeo et al.

    An element of the transforming frowth factor-β1 5′-untranslated region represses translation and specifically binds a cytosolic factor

    Mol. Endocrinol.

    (1993)
  • U.K. Laemmli

    Cleavage of structural proteins during the assembly of the head of bacteriophage T4

    Nature

    (1970)
  • H.P. Rodemann et al.

    Selective enrichment and biochemical characterisation of seven human skin fibroblasts cell types in vitro

    Exp. Cell. Res.

    (1989)
  • H.Y. Zhang et al.

    Lung fibroblast alpha smooth muscle actin expression and contractile phenotype in bleomycin-induced pulmonary fibrosis

    Am. J. Pathol.

    (1996)
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