Regular articleTGF-β1-mediated fibroblast–myofibroblast terminal differentiation—the role of 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.
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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.
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