Smooth Muscle Cell Tissue Research Articles

Expression and functional activity of four myocardin isoforms

Myocardin (MYOCD) is an essential component of a molecular switch for the expression of contractile genes in smooth muscle and cardiac muscle cells. The Myocd gene comprises at least fifteen exons, including two alternately spliced exons designated 2a and 10a. We investigated tissue-specific Myocd expression in mouse, rat and human tissues to determine the conservation in expression of each Myocd splice variant and to ascertain whether any functional differences exist among MYOCD isoforms. Conventional and quantitative RT-PCR revealed the dominant expression of Myocd exon 2a ( Myocd_v3) in smooth muscle cell (SMC)-rich tissues (aorta and bladder) with little expression in heart across all species studied. Each species of heart showed primarily a longer version of Myocd ( Myocd_v1) without exon 2a. While exclusion of exon 2a was common in all cardiac muscle samples, exon skipping of Myocd exon 10a was a rare event in both cardiac muscle and SMC tissues. In general, all four MYOCD isoforms showed comparable stimulation of SMC promoters. On the other hand, Myocd_v1 and Myocd_v2 were more active than Myocd_v3 and Myocd_v4 in stimulating cardiac muscle promoters and Myocd_v1's activity was augmented in the presence of the cardiac transcription factor, MEF2C. Importantly, whereas all four MYOCD isoforms similarly induced expression of endogenous SMC genes in a prostate tumor cell line (LNCaP), none could induce the endogenous expression of specific cardiac markers. These results are the first to show relative expression and activities of the major myocardin isoforms across disparate species. We propose a new myocardin nomenclature reflecting the dominant splice variants expressed in cardiac muscle ( Myocd_v1 and v2) versus SMC-rich tissues ( Myocd_v3 and v4).

Kruppel-like Factor 4 Abrogates Myocardin-induced Activation of Smooth Muscle Gene Expression

Platelet-derived growth factor BB (PDGF-BB) has been shown to be an extremely potent negative regulator of smooth muscle cell (SMC) differentiation. Moreover, previous studies have demonstrated that the Kruppel-like transcription factor (KLF) 4 potently represses the expression of multiple SMC genes. However, the mechanisms whereby KLF4 suppresses SMC gene expression are not known, nor is it clear whether KLF4 contributes to PDGF-BB-induced down-regulation of SMC genes. The goals of the present studies were to determine the molecular mechanisms by which KLF4 represses expression of SMC genes and whether it contributes to PDGF-BB-induced suppression of these genes. Results demonstrated that KLF4 markedly repressed both myocardin-induced activation of SMC genes and expression of myocardin. KLF4 was rapidly up-regulated in PDGF-BB-treated, cultured SMC, and a small interfering RNA to KLF4 partially blocked PDGF-BB-induced SMC gene repression. Both PDGF-BB and KLF4 markedly reduced serum response factor binding to CArG containing regions within intact chromatin. Finally, KLF4, which is normally not expressed in differentiated SMC in vivo, was rapidly up-regulated in vivo in response to vascular injury. Taken together, results indicate that KLF4 represses SMC genes by both down-regulating myocardin expression and preventing serum response factor/myocardin from associating with SMC gene promoters, and suggest that KLF4 may be a key effector of PDGF-BB and injury-induced phenotypic switching of SMC.

Smooth muscle-specific expression of the smooth muscle myosin heavy chain gene in transgenic mice requires 5'-flanking and first intronic DNA sequence.

The smooth muscle myosin heavy chain (SM-MHC) gene encodes a major contractile protein whose expression exclusively marks the smooth muscle cell (SMC) lineage. To better understand smooth muscle differentiation at the transcriptional level, we have initiated studies to identify those DNA sequences critical for expression of the SM-MHC gene. Here we report the identification of an SM-MHC promoter-intronic DNA fragment that directs smooth muscle-specific expression in transgenic mice. Transgenic mice harboring an SM-MHC-lacZ reporter construct containing approximately 16 kb of the SM-MHC genomic region from -4.2 to + 11.6 kb (within the first intron) expressed the lacZ transgene in all smooth muscle tissue types. The inclusion of the intronic sequence was required for transgene expression, since 4.2 kb of the 5'-flanking region alone was not sufficient for expression. In the adult mouse, transgene expression was observed in both arterial and venous smooth muscle, in airway smooth muscle of the trachea and bronchi, and in the smooth muscle layers of all abdominal organs, including the stomach, intestine, ureters, and bladder. During development, transgene expression was first detected in airway SMCs at embryonic day 12.5 and in vascular and visceral SMC tissues by embryonic day 14.5. Of interest, expression of the SM-MHC transgene was markedly reduced or absent in some SMC tissues, including the pulmonary circulation. Moreover, the transgene exhibited a heterogeneous pattern between individual SMCs within a given tissue, suggesting the possibility of the existence of different SM-MHC gene regulatory programs between SMC subpopulations and/or of episodic rather than continuous expression of the SM-MHC gene. To our knowledge, results of these studies are the first to identify a promoter region that confers complete SMC specificity in vivo, thus providing a system with which to define SMC-specific transcriptional regulatory mechanisms and to design vectors for SMC-specific gene targeting.

Overexpression of insulin-like growth factor-binding protein-4 (IGFBP-4) in smooth muscle cells of transgenic mice through a smooth muscle alpha-actin-IGFBP-4 fusion gene induces smooth muscle hypoplasia.

Insulin-like growth factor I (IGF-I) has been postulated to function as a smooth muscle cell (SMC) mitogen and to play a role in the pathogenesis of bladder hypertrophy, estrogen-induced uterine growth, and restenosis after arterial angioplasty. IGF-binding protein-4 (IGFBP-4) inhibits IGF-I action in vitro and is the most abundant IGFBP in the rodent arterial wall. To explore the function of this binding protein in vivo, transgenic mouse lines were developed harboring fusion genes consisting of a rat IGFBP-4 complementary DNA cloned downstream of either a -724 bp fragment of the mouse smooth muscle alpha-actin 5'-flanking region (SMP2-BP-4) or -1074 bp, 63 bp of 5'-untranslated region, and 2.5 kb of intron 1 of smooth muscle alpha-actin (SMP8-BP-4). SMP2-BP-4 mice expressed low levels of the exogenous IGFBP-4 messenger RNA (mRNA), which was not specifically targeted to SMC-rich tissue environments, and were therefore not analyzed further. Six SMP8-BP-4 transgenic lines derived from separate founders were characterized. Mating of hemizygous SMP8-BP-4 mice with controls produced about 50% transgenic offspring, with equal sex distribution. Expression of IGFBP-4 mRNA in nontransgenic littermates was maximal in liver and kidney. By contrast, transgenic IGFBP-4 mRNA expression, distinguished because of a smaller transcript size, was confined to SMC-containing tissues, with the following hierarchy: bladder > aorta > stomach = uterus. There was no transgene expression in skeletal muscle, brain, or cardiac myocytes. The abundance of IGFBP-4 measured by Western ligand blotting or by immunoblotting, was 8- to 10-fold higher in aorta and bladder of SMP8-BP-4 mice than in their nontransgenic littermates, with no change in plasma IGFBP-4 levels. Transgenic mice exhibited a significant reduction in wet weight of SMC-rich tissues, including bladder, intestine, aorta, uterus, and stomach, with no change in total body or carcass weight. In situ hybridization showed that transgene expression was targeted exclusively to the muscular layers of the arteries, veins, bladder, ureter, stomach, intestine, and uterus. Overexpression of IGFBP-4 was associated with SMC hypoplasia, a reciprocal phenotype to that of transgenic mice overexpressing IGF-I under control of the same promoter (SMP8-IGF-I). Double transgenic mice derived from mating SMP8-BP-4 with SMP8-IGF-I animals showed a modest decrease in wet weight at selected SMC tissues. Although we cannot exclude that the effects of IGFBP-4 may be IGF independent, these data suggest that IGFBP-4 is a functional antagonist of IGF-I action on SMC in vivo.

Phosphorothioate Oligodeoxynucleotide Inhibition of Restenosis.

Antisense therapy with phosphorothioate oligodeoxynucleotides (PS oligos) has emerged as a potentially useful strategy for inhibiting angioplasty restenosis. Several groups have reported that PS oligos inhibit in vitro vascular smooth muscle cell (SMC) proliferation as well as in vivo neointimal formation after rat carotid artery balloon injury. More recent studies have revealed the presence of a PS oligo G-quartet inhibitory effect, reflecting binding of oligonucleotides to cellular proteins, which is distinct from a hybridization-dependent antisense effect. Studies with the 28-mer phosphorothioate cytidine homopolymer S-dC28 have demonstrated the presence of a non-G-quartet, non-sequence-specific inhibitory effect on SMC proliferation and migration in vitro and neointimal hyperplasia after rat carotid balloon injury in vivo. These effects are the result, in part, of the avid binding of the polyanion PS oligos to heparin-binding growth factors, such as platelet-derived growth factor (PDGF) and basic fibroblast growth factor. Moreover, S-dC28 attenuates PDGF-induced SMC tissue plasminogen activator antigen levels without affecting SMC plasminogen activator inhibitor-1 levels, thereby suggesting a PS oligo net antifibrinolytic effect that would impede SMC migration. Therefore, the further development of these drugs to inhibit angioplasty restenosis must consider the hybridization-specific antisense effects, the non-G-quartet inhibitory effects, and the non-G-quartet, non-sequence-specific inhibitory effects of the pleiotropic PS oligos.

Expression of insulin-like growth factors (IGFs), their receptors and IGF binding protein-3 in normal, benign and malignant smooth muscle tissues.

To assess the role of insulin-like growth factors (IGFs) in growth and transformation of normal (myometrium) and tumorous smooth muscle cell (SMC) tissues, in situ hybridization (ISH) analysis for insulin-like growth factor I and II (IGF-I and IGF-II) mRNAs was combined with detection of IGF peptides, their receptors and IGF binding protein-3 (IGFBP-3). mRNAs for both IGFs were detected in smooth muscle cells in normal, benign and malignant SMC tissues, together with the IGF peptides, both IGF receptors and IGFBP-3. This suggests an autocrine role for both IGFs. Leiomyomas had higher IGF-I peptide levels and higher levels of type I IGF receptors than myometrium, supporting the idea that IGFs play a role in the growth and transformation of these tumours. Low-grade leiomyosarcomas contained more IGF-II mRNAs than myometrium and leiomyoma, fewer type II IGF/mannose 6-phosphate receptors and less IGFBP-3 than myometrium and, in addition, fewer IGF-I mRNAs and type I IGF receptors than leiomyoma. Intermediate- and high-grade leiomyosarcomas had intermediate levels of IGF-II mRNAs and peptide, ranging between those in myometrium and low-grade leiomyosarcomas. Thus, growth and transformation of leiomyosarcomas may be regulated by IGF-II, although more markedly in low-grade than in high-grade leiomyosarcomas. In conclusion, the various categories of SMC tissues are associated with a distinct expression pattern of the IGF system. This suggests that each category of SMC tumours arises as a distinct entity and that there is no progression of transformation in these tissues.