Coordinate Expression of α-Tropomyosin and Caldesmon Isoforms in Association with Phenotypic Modulation of Smooth Muscle Cells

Abstract

Isoform diversity of tropomyosin is generated from the limited genes by a combination of differential transcription and alternative splicing. In the case of the ␣-tropomyosin (␣-TM) gene, exon 2a rather than exon 2b is specifically spliced in ␣-TM-SM mRNA, which is one of the major tropomyosin isoforms in smooth muscle cells. Here we demonstrate that expressions of ␣-tropomyosin and caldesmon isoforms are coordinately regulated in association with phenotypic modulation of smooth muscle cells. Molecular cloning and Western and Northern blottings have revealed that in addition to the downregulation of ␤-TM-SM, ␣-TM-SM converted to ␣-TM-F1 and ␣-TM-F2 by a selectional change from exon 2a to exon 2b during dedifferentiation of smooth muscle cells in culture. Simultaneously, a change of caldesmon isoforms from high M r type to low M r type was also observed by alternative selection between exons 3b and 4 in the caldesmon gene during this process. In contrast, cultured smooth muscle cells maintaining a differentiated phenotype continued to express ␣-TM-SM, ␤-TM-SM, and high M r caldesmon. In situ hybridization revealed specific coexpression of ␣-TM-SM and high M r caldesmon in smooth muscle in developing embryos. These results suggest a common splicing mechanism for phenotype-dependent expression of tropomyosin and caldesmon isoforms in both visceral and vascular smooth muscle cells.It is important to elucidate the molecular mechanism of phenotypic modulation of smooth muscle cells (SMCs) 1 such as vasculogenesis, enterogenesis, atherosclerosis, hypertension, and leiomyogenic tumorigenesis. The SMCs are derived from mesodermal precursors, but the intracellular and extracellular factors determining the SMC lineage and its phenotype remain unclear. The search for molecular parameters indicating SMC phenotype is a first step in analyzing phenotypic modulation of SMCs. Several cytoskeletal and contractile proteins are such candidates. Among them, changes of actin (1, 2), caldesmon (CaD) (1, 3, 4), myosin heavy chain (5, 6), and vinculin/metavinculin (1, 7) isoforms are closely associated with phenotypic modulation of SMCs. Recent studies have focused on the gene regulation of such parameters (8 -14). In addition to these isoform changes, expression of ␣-smooth muscle actin (␣-SM actin) (15, 16), CaD (1, 3, 4), myosin heavy and light chains (5, 6, 17), meta-vinculin (1, 7), SM22, and calponin (18 -20) have been reported to be up-regulated during differentiation of SMCs, but down-regulated during dedifferentiation, suggesting the involvement of SMC phenotype-dependent transcriptional regulation in the SMC-specific parameter genes. In fact, the transcriptional machineries of ␣-SM actin (8, 9), CaD (12), myosin heavy chain (13), SM22 (21), and calponin (22) have been partially characterized.Tropomyosin (TM) is a predominant helical protein that binds to actin groves. Recent evidence suggests that Ca 2ϩ -dependent actin-myosin interaction in smooth and nonmuscle cells is controlled by myosin-and actin-linked dual regulation....

Changes in Ca2+ Signaling and Contractile Protein Isoforms in Smooth Muscle Cells from Guinea Pig Ileum during Culture.

“…Although more detailed studies are required, our results suggested that the 41 kDa tropomyosin monomer plays an important role in contraction. Regarding the interaction between tropomyosin and caldesmon, coincident expression of α-tropomyosin and caldesmon isoforms in SMCs has been reported (Kashiwada et al, 1997). While the present result on tropomyosin is similar, studies at the mRNA level and on the relationship between caldesmon isoforms are required before the role of tropomyosin isoforms can be understood.…”

“…If culture conditions were achieved which kept the SMCs in their differentiated state as reported for other SMCs (Bowers and Dahm, 1993;Kashiwada et al, 1997;Ma et al, 1998;Halayko et al, 1999), protein isoform changes during culture may be expected to be different from those in this study. The differences would provide important information about the role of each isoform of the contractile proteins and of the contraction mechanism of SMCs.…”

Transcriptional Activation of β-Tropomyosin Mediated by Serum Response Factor and a Novel Barx Homologue, Barx1b, in Smooth Muscle Cells

“…as SMC-differentiation molecular markers (21). The chicken ␤-TM gene contains muscle and non-muscle promoters, and both smooth (␤-TMsm) and skeletal (␤-TMsk) muscle isoforms are transcribed from the same muscle promoter (22).…”

Caldesmon and the Regulation of Cytoskeletal Functions

“…20,21 Although a number of smooth muscle specific proteins undergo such a differentiationdependent isoform switchover, including actin, 22 myosin heavy chain, 23 calponin, 24 vinculin 25 and Tm, 26 the expression of CaD and Tm are probably most closely related to each other. In fact, the same splicing machinery appears to work on both CaD and Tm, as the two α-Tm isoforms (α-Tm-SM or Tm6 and α-Tm-F1/2 or Tm2; for Tm nomenclature, see Gunning et al 27 ) are expressed in a tightly coordinated fashion with the two isoforms of CaD both in vivo and in vitro.…”

“…In fact, the same splicing machinery appears to work on both CaD and Tm, as the two α-Tm isoforms (α-Tm-SM or Tm6 and α-Tm-F1/2 or Tm2; for Tm nomenclature, see Gunning et al 27 ) are expressed in a tightly coordinated fashion with the two isoforms of CaD both in vivo and in vitro. 26,28 Interestingly, when cells were forced to express Tm1 but not Tm2, there was also an up-regulation of CaD expression. 29 It has been shown that a serum response factor (SRF) is necessary but not sufficient to transactivate the CaD promoter.…”