Stretch induces lung embryonic mesenchymal cells to follow a myogenic pathway. Using this system we identified a set of stretch-responsive factors, which we referred to as TIPs (tension-induced/inhibited proteins). TIPs displayed signature motifs characteristic of nuclear receptor coregulators and chromatin remodeling enzymes. A genomic BLAST search suggested that the three TIPs identified were isoforms originated by alternative splicing from a single gene. Functional studies revealed that TIP-1 and TIP-3 were involved in the cell's selection of the myogenic or the adipogenic pathway. TIP-1, induced by stretch, promoted myogenesis, while TIP-3, inhibited by stretch, stimulated adipogenesis. The selection involved TIP-mediated chromatin remodeling via a histone acetylation process and depended on TIP-1 and TIP-3 nuclear receptor binding boxes (NRBs). This study, therefore, suggests a new developmental mechanism linking the presence or absence of tension with divergent differentiation pathways.
Round embryonic mesenchymal cells have the potential to differentiate into smooth muscle (SM) cells upon spreading/elongation (Yang, Y., K.C. Palmer, N. Relan, C. Diglio, and L. Schuger. 1998. Development. 125:2621–2629; Yang, Y., N.K. Relan, D.A. Przywara, and L. Schuger. 1999. Development. 126:3027–3033; Yang, Y., S. Beqaj, P. Kemp, I. Ariel, and L. Schuger. 2000. J. Clin. Invest. 106:1321–1330). In the developing lung, this process is stimulated by peribronchial accumulation of laminin (LN)-2 (Relan, N.K., Y. Yang, S. Beqaj, J.H. Miner, and L. Schuger. 1999. J. Cell Biol. 147:1341–1350). Here we show that LN-2 stimulates bronchial myogenesis by down-regulating RhoA activity. Immunohistochemistry, immunoblotting, and reverse transcriptase–PCR indicated that RhoA, a small GTPase signaling protein, is abundant in undifferentiated embryonic mesenchymal cells and that its levels decrease along with SM myogenesis. Functional studies using agonists and antagonists of RhoA activation and dominant positive and negative plasmid constructs demonstrated that high RhoA activity was required to maintain the round undifferentiated mesenchymal cell phenotype. This was in part achieved by restricting the localization of the myogenic transcription factor serum response factor (SRF) mostly to the mesenchymal cell cytoplasm. Upon spreading on LN-2 but not on other main components of the extracellular matrix, the activity and level of RhoA decreased rapidly, resulting in translocation of SRF to the nucleus. Both cell elongation and SRF translocation were prevented by overexpression of dominant positive RhoA. Once the cells underwent SM differentiation, up-regulation of RhoA activity induced rather than inhibited SM gene expression. Therefore, our studies suggest a novel mechanism whereby LN-2 and RhoA modulate SM myogenesis.
P311, also called PTZ17, was identified by suppressive subtraction hybridization as potentially involved in smooth muscle (SM) myogenesis. P311 is an 8-kDa protein with several PEST-like motifs found in neurons and muscle. P311 transfection into two fibroblast cell lines, NIH 3T3 and C3H10 T1/2, induced phenotypic changes consistent with myofibroblast transformation, including upregulation of SM α−actin and SM22, induction of FGF-2, VEGF, PDGF, and PDGF receptors, upregulation of integrins α3 and α5, and increased proliferation rate. The P311-mediated changes differed, however, from the well-characterized myofibroblast in that P311 inhibited TGF-β1, TGF-β receptor 2, and TGF-β1–activating MMP-2 and MMP-9, with the resultant decrease in collagen 1 and 3 expression. The effect of P311 on collagen was overcome by exogenous TGF-β1, indicating that the cells were responsive to TGF-β1 paracrine stimulus. In support of a role for P311 in vivo, immunohistochemical examination of human wounds showed P311 only in myofibroblasts and their activated precursors. To our knowledge, these studies are the first to implicate P311 in myofibroblast transformation, to demonstrate that transformation may occur independently of TGF-β1, and to suggest that P311 may prevent fibrosis
Pulmonary lymphangioleiomyomatosis (LAM) is characterized by abnormal smooth muscle-like cell proliferation leading to tissue destruction and cyst formation. We demonstrate that serum response factor (SRF), a critical smooth muscle transcription factor, is overexpressed in LAM cells. To determine whether abnormal SRF levels might have a pathogenic role in LAM, we transfected SRF into mouse lung fibroblasts and performed a cDNA array analysis. High SRF level upregulated the expression of matrix metalloproteinase (MMP)-2 and MMP-14, two MMPs previously shown to be increased in LAM. In addition, SRF down-regulated tissue inhibitor of metalloproteinase (TIMP)-3, one of their inhibitors. TIMP-3 inhibition was further confirmed by reverse transcriptase/polymerase chain reaction, immunoblotting, and immunostaining of human lung fibroblasts transfected with SRF fused to DsRed2 (a red variant of green fluorescent protein). To determine the in vivo significance of our findings, we immunostained 12 LAM cases for TIMP-3. In eight of them, TIMP-3 was ubiquitously present in normal lung parenchyma, but it was absent in LAM lesions. In the remaining cases, including two out of five normal control lungs, the antibody immunoreacted exclusively with elastin, probably due to suboptimal tissue processing. Because timp-3-null mice develop spontaneous emphysema, our findings suggest that SRF-mediated TIMP-3 inhibition might contribute to the tissue damage seen in LAM.
P311, also called PTZ17, was identified by suppressive subtraction hybridization as potentially involved in smooth muscle (SM) myogenesis. P311 is an 8-kDa protein with several PEST-like motifs found in neurons and muscle. P311 transfection into two fibroblast cell lines, NIH 3T3 and C3H10 T1/2, induced phenotypic changes consistent with myofibroblast transformation, including upregulation of SM α−actin and SM22, induction of FGF-2, VEGF, PDGF, and PDGF receptors, upregulation of integrins α3 and α5, and increased proliferation rate. The P311-mediated changes differed, however, from the well-characterized myofibroblast in that P311 inhibited TGF-β1, TGF-β receptor 2, and TGF-β1-activating MMP-2 and MMP-9, with the resultant decrease in collagen 1 and 3 expression. The effect of P311 on collagen was overcome by exogenous TGF-β1, indicating that the cells were responsive to TGF-β1 paracrine stimulus. In support of a role for P311 in vivo, immunohistochemical examination of human wounds showed P311 only in myofibroblasts and their activated precursors. To our knowledge, these studies are the first to implicate P311 in myofibroblast transformation, to demonstrate that transformation may occur independently of TGF-β1, and to suggest that P311 may prevent fibrosis.
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