Pulmonary myofibroblasts are implicated in various pulmonary interstitial diseases. This work focuses on delineating the expression pattern, mechanism of regulation, and function of skeletal MyHC within the context of the pulmonary myofibroblast. Whether these pathways regulate MyHC expression in myofibroblasts is addressed in the current study. CaM kinase can regulate transcription by phosphorylation of myocyte enhancer factor 2 (MEF2), resulting in nuclear export of the repressor protein histone deacetylase ( Lu et al., 2000 McKinsey et al., 2000). Calcineurin dephosphorylates the nuclear factor of activated T cells (NFAT) transcription factors, allowing their nuclear transport ( Chin et al., 1998). The calcineurin and calmodulin (CaM) kinase pathways have been shown to differentially regulate MyHC isoform expression in muscle cells ( Allen et al., 2001 Allen and Leinwand, 2002). Although no evidence exists for direct regulation of skeletal MyHC genes by MRFs, MyoD indirectly regulates the transcriptional activity of the skeletal MyHC IIb gene ( Takeda et al., 1995 Lakich et al., 1998 Wheeler et al., 1999). MRFs can activate the entire skeletal muscle gene program when expressed exogenously in a number of cell types ( Weintraub et al., 1989). Active myogenic regulatory factors (MRFs), a family of skeletal muscle–specific transcription factors, are expressed in a number of myofibroblasts ( Rockey et al., 1993 Mayer and Leinwand, 1997 Kaminski et al., 2000). What factors regulate skeletal muscle gene expression, specifically MyHC expression, in these cells is also unclear. Myofibroblasts have the unusual property of expressing not only smooth muscle proteins, but also a number of sarcomeric proteins otherwise found only in skeletal muscle, including several skeletal-specific myosin heavy chain (MyHC) isoforms ( Mayer and Leinwand, 1997 van der Ven and Furst, 1998). Indeed, ASMA has been implicated in the contractility of dermal, gingival, periodontal ligament, and pulmonary myofibroblasts ( Arora and McCulloch, 1994 Germain et al., 1994 Hinz et al., 2001), and inhibition of ASMA expression blocks chicken embryonic myofibroblast contraction ( Feugate et al., 2002).
Other characteristic properties include the secretion of ECM and contractility (for reviews see Serini and Gabbiani, 1999 Tomasek et al., 2002). The expression of α smooth muscle actin (ASMA) and various intermediate filaments, e.g., desmin and vimentin, is most frequently used to identify myofibroblasts. In vivo, myofibroblasts are thought to arise from the transient differentiation of resident fibroblasts through multiple paracrine-mediated pathways including TGFβ mechanisms (for reviews see Desmouliere and Gabbiani, 1996 Vaughan et al., 2000). However, the persistence of these cells after injury and their continued secretion of ECM have implicated them in various fibroproliferative processes, including fibrotic diseases of the liver, kidney, and lung ( Hautekeete and Geerts, 1997 Powell et al., 1999 Badid et al., 2001 Phan 2002). They are essential for the formation of functional adult tissues and are intimately involved in tissue homeostasis (for reviews see Desmouliere and Gabbiani, 1996 Powell et al., 1999 Walker et al., 2001). Myofibroblasts are unique cells, possessing ultrastructural characteristics of both muscle and nonmuscle cells. Interestingly, the regulation of skeletal myosin expression in myofibroblasts is distinct from that observed in muscle cells and suggests that cell context is important in its control. To understand the molecular mechanisms whereby skeletal muscle genes are regulated in myofibroblasts, we have found that members of the myogenic regulatory factor family of transcription factors and Ca 2+-regulated pathways are involved in skeletal MyHC promoter activity.
Furthermore, inhibition of skeletal myosin activity and myofibroblast contraction results in a decrease in both ASMA and skeletal MyHC promoter activity and ASMA protein expression, suggesting a potential coupling of skeletal myosin activity and ASMA expression in myofibroblast differentiation. In this study, we show that pulmonary myofibroblasts express three of the eight known sarcomeric myosin heavy chains (MyHCs) (IIa, IId, and embryonic) and that skeletal muscle myosin enzymatic activity is required for pulmonary myofibroblast contractility. Typically myofibroblasts are identified by the expression of α smooth muscle actin (ASMA) however some myofibroblasts also express sarcomeric proteins.
Myofibroblasts are unique contractile cells with both muscle and nonmuscle properties.