Transforming growth factor-β (TGF-β) type I and type II receptors are both required for TGF-β-mediated extracellular matrix production in lung fibroblasts

Abstract

Transforming growth factor-β (TGF-β) regulates a variety of cellular activities including cell growth, differentiation and extracellular matrix production. The TGF-β type I and type II serine/threonine kinase receptors (TβRI and TβRII) have been identified as signal-transducing TGF-β receptors. This study was undertaken to examine the role of the type I and type II receptors in TGF-β-induced extracellular matrix production of lung fibroblasts. We constructed expression plasmids containing truncated derivatives of TβRI and TβRII that lacked the cytoplasmic serine/threonine kinase domain (TβRIΔK and TβRIIΔK), and transfected them into lung fibroblasts. TβRIIΔK expressed by lung fibroblasts was able to bind ¹²⁵I-TGF-β1, whereas TβRIΔK was unable to bind ligand when expressed alone. Co-expression with TβRII was required for binding and cross-linking of TGF-β1 to TβRIΔK. Lung fibroblasts upregulate tenascin and fibronectin production when treated with TGF-β1. The kinase-defective deletions of both TβRI and TβRII were dominant-acting inhibitors of TGF-β signal transduction. Expression of either TβRIΔK or TβRIIΔK alone was sufficient to block TGF-β-induced tenascin and fibronectin production of lung fibroblasts. The results indicate that both TβRI and TβRII were required for TGF-β signaling in regulation of extracellular matrix production by lung fibroblasts.

Introduction

Transforming growth factor-β (TGF-β) belongs to a large family of structurally related cytokines that has multiple effects on a wide variety of cell types (Massague, 1990). These effects include modulation of growth and regulation of gene transcription. TGF-β elicits its biological effects on cells through binding to cell surface transmembrane receptors. A number of different types of putative receptors for TGF-β, including three distinct size classes termed type I (TβRI, 50–60 kDa), type II (TβRII, 75–85 kDa) and type III (TβRIII, a 280 kDa proteoglycan with a 120 kDa core protein), have been identified by affinity cross-linking experiments (Cheifetz and Massague, 1989, Massague, 1992). Sequence analysis of TβRIII revealed that it is a transmembrane proteoglycan with a short and highly conserved cytoplasmic domain that has no apparent signal motif (Lopez-Casillas et al., 1991, Wang et al., 1991). Both type I and type II receptors are transmembrane serine-threonine kinases that are indispensable for TGF-β signaling (Lin et al., 1992, Attisano et al., 1993, Franzen et al., 1993). A ligand-induced heterodimer model was proposed for TGF-β signal transduction (Wrana et al., 1994, Vivien et al., 1995). TGF-β binds directly to TβRII, which then recruits TβRI to form a heteromeric complex. Activated TβRII transphosphorylates the type I receptor kinase, thereby activating TβRI, which then propagates the signal to downstream substrates. Recent studies revealed that Mothers against dpp (Mad) in Drosophila and its homologs play important roles in the intracellular signal transduction of the serine/threonine kinase receptors (Derynck and Zhang, 1996, Kretzschmar and Massague, 1998). TGF-β induces heteromeric complexes of Smads 2, 3 and 4, and their concomitant translocation to the nucleus, which is required for efficient TGF-β signal transduction (Massague, 1996, Nakao et al., 1997).

TGF-β stimulates extracellular matrix synthesis that has been implicated in embryogenesis, wound healing, and fibroproliferative responses to tissue injury (McGowan, 1992). Lung fibroblasts are target cells for TGF-β and they are the primary sources of extracellular matrix in the lung. TGF-β has been found to induce tenascin in rat fetal lung explant culture (Zhao and Young, 1995a). TGF-β stimulates the expression of fibronectin and both subunits of the fibronectin receptor by cultured human lung fibroblasts (Roberts et al., 1988), TGF-β increases the synthesis of type I, III and V collagens in human lung fibroblasts (Raghu et al., 1989, Fine et al., 1990), and elastin in rat lung fibroblasts (McGowan and McNamer, 1990, McGowan et al., 1997). However, the functional significance of the TGF-β type I and type II receptors in TGF-β-induced extracellular matrix production has not been fully elucidated. Molecular and cytogenetic analyses suggest two models for TGF-β signal transduction. One model proposes that both TβRI and TβRII are required for regulation of cellular growth and extracellular matrix production by TGF-β (Laiho et al., 1990, Laiho et al., 1991, Wieser et al., 1993), whereas the other indicates that the effect of TGF-β on cell growth is mediated by TβRII and the effect of the growth factor on extracellular matrix synthesis is dependent upon TβRI (Geiser et al., 1992, Chen et al., 1993, Ebner et al., 1993).

To gain insight into the role of TβRI and TβRII in TGF-β-mediated induction of extracellular matrix, we constructed expression plasmids containing rat TβRI and TβRII cDNAs that lacked the cytoplasmic serine/threonine kinase domain (TβRIΔK and TβRIIΔK), and transfected them into rat lung fibroblast cells. Overexpression of the dominant negative TβRI or TβRII mutant blocked TGF-β-induced tenascin and fibronectin production by lung fibroblasts. The present data thus indicate that both the type I and type II receptors are required for TGF-β signaling that modulates the extracellular matrix production of lung fibroblasts.