Smooth Muscle Gamma-actin Research Articles

New concepts concerning prostate cancer screening

Prostate Cancer (CaP) is rapidly becoming a worldwide health issue. While CaP mortality has decreased in recent years, coincident with the widespread use of Prostate-Specific Antigen (PSA) screening, it remains the most common solid tumor in men and is the second leading cause of cancer death in the United States. The frequency of CaP is growing not only in western cultures, but also its incidence is dramatically increasing in eastern nations. Recently, examination of data from long-term trials and follow up has cast a shadow on the effectiveness of employing PSA as a primary screening tool for CaP. In this review, we not only summarize opinions from this examination and synthesize recommendations from several groups that suggest strategies for utilizing PSA as a tool, but also call for research into biomarkers for CaP diagnosis and disease progression. We also describe our recent work that identified a smooth muscle contractile protein in prostate epithelia, namely smooth muscle gamma actin, and indicate the potential for this molecule as a new unique footprint and as a CaP marker.

Expression of genes encoding smooth muscle contractile proteins in vaginal tissue of women with and without pelvic organ prolapse

We hypothesize that the expression of genes encoding vaginal smooth muscle (SM) contractile proteins is altered in patients with pelvic organ prolapse (POP) and is influenced by age and menopausal status. We aim to analyze the expression of SM-myosin heavy chain (MHY11), caldesmon (CALD1), SM gamma-actin (ACTG2), and tropomyosin (TPM1), in premenopausal and postmenopausal women with advanced POP and asymptomatic controls. During total hysterectomy we collected anterior vaginal wall biopsy samples from 55 women, 37 premenopausal (23 patients and 14 controls), and 18 postmenopausal women (13 patients and 5 controls). Total mRNA from the tissues was quantified by real-time RT-PCR. MHY11 gene expression was down-regulated in premenopausal POP patients compared to premenopausal controls (fivefold, P = 0.002). In the postmenopausal groups, we observed a sixfold increase in the CALD1 gene expression in POP patients compared to asymptomatic controls (P = 0.03). The gene expression of CALD1, ACTG2, and TPM1 was significantly down-regulated in vaginal tissue of healthy women after menopause (P < 0.05). Dysregulation of the vaginal SM content in POP patients involves alteration of different cellular pathways according to age and menopausal status.

Expression of Subtype-Specific Group 1 Leiomyosarcoma Markers in a Wide Variety of Sarcomas by Gene Expression Analysis and Immunohistochemistry

Leiomyosarcomas (LMSs) constitute approximately one quarter of all sarcomas and are usually defined by morphologic criteria and/or immunoreactivity for actin or desmin. Among high-grade lesions, the distinction from undifferentiated pleomorphic sarcoma (UPS) can be problematic, and previous studies have shown that a significant number of LMS cases may be hiding under the diagnosis of UPS. We recently described 3 novel molecular LMS subtypes that are distributed similarly over LMSs of gyneocologic and non-gyneocologic origins. The group 1 subtype shows an improved disease-specific survival compared with the other 2 groups that is independent of histologic grade. Group 1 comprises approximately 25% of all LMSs, and is defined by a shared pattern of gene expression, a distinct pattern of genomic changes, and reactivity for at least 3 of 5 immunohistochemistry (IHC) markers (smooth muscle gamma actin, calsequestrin 2, human muscle cofilin2, myosin light chain kinase, and sarcolemmal membrane associated protein), as tested on 271 cases of LMS in tissue microarrays. These IHC markers have not been well characterized in non-LMS sarcomas. Here we provide a characterization of these 5 markers across normal tissues, an additional 59 cases of LMS, and a wide range of 565 non-LMS soft tissue tumors from 44 diagnostic categories, with a focus on UPS. When analyzed individually, the 5 markers were found to be expressed in many sarcomas other than LMSs. However, when analyzed by the same criteria used for the recognition of group 1 LMSs, in which a case is scored positive when at least 3 of 5 markers reacted, coordinate expression was seen in significant numbers of cases from only 3 diagnostic groups that included 22% of leiomyomas (n=22), 16% of gastrointestinal stromal tumors (n=43), and 18% of endometrial stromal sarcomas (n=11). In addition, 5% (n=57) of UPSs showed a staining pattern similar to that seen in group 1 LMSs. To further examine the possibility that group 1 LMS constitutes a small part of cases diagnosed as UPS, we examined the expression of the top 500 genes from the group 1 LMS expression signature in 29 UPSs by complementary DNA microarray. Unsupervised hierarchical clustering of 29 UPS expression showed that 2 (7%) had coordinated high levels of expression of genes from the group 1 LMS signature, a rate similar to that seen by IHC analysis. These findings show that group 1 LMS IHC markers smooth muscle gamma actin, calsequestrin 2, human muscle cofilin2, myosin light chain kinase, and sarcolemmal membrane associated protein when coordinately expressed have specificity for a subset of LMS when compared with other sarcomas, and may be useful for the recognition of group 1 LMS cases within cases diagnosed as UPS.

Proteomic analysis of pubocervical fascia in women with and without pelvic organ prolapse and urodynamic stress incontinence

This pilot study compares protein expression patterns in the pubocervical fascia of women with pelvic organ prolapse (POP) and stress urinary incontinence (SUI) and asymptomatic women with normal pelvic support. Samples of pubocervical fascia were collected from four women with POP and SUI and from three asymptomatic (control) women. These were analyzed using two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The expression levels of transgelin, smooth muscle gamma-actin, myosin light polypeptide 6, and alpha-1 antitrypsin precursor were more than twofold higher in patients than in controls. An additional five proteins were overexpressed (more than twofold) in patients, while three proteins were detected only in the patients. This pilot study is the first study to estimate changes in protein expression in the pubocervical fascia of human patients with POP. These changes could be related to the pathophysiology of POP.

Smooth Muscle-Specific Gene Delivery in the Vasculature Based on Restriction of DNA Nuclear Import

The two currently employed approaches restricting gene delivery and/or expression to desired cell types in vivo rely on cell surface targeting or cell-specific promoters. We have developed a third approach based on cell-specific nuclear transport of the delivered plasmid DNA. We have previously shown that plasmid nuclear import in non-dividing cells is sequence-specific and have identified a set of cell-specific DNA nuclear targeting sequences that can be used to limit DNA nuclear import to desired cell types. Specifically we have identified elements of the smooth muscle gamma actin (SMGA) promoter that direct plasmid nuclear import selectively in smooth muscle cells (SMCs) in vitro (Vacik et al, 1999, Gene Therapy 6:1006-1014). In the present study, we demonstrate that the SMC-specific DNA nuclear targeting sequence from the SMGA promoter drives nuclear accumulation of plasmids and subsequent gene expression exclusively in the smooth muscle cell layer of the vessel wall in the intact vasculature of rats using electroporation mediated delivery. These results demonstrate that certain DNA nuclear targeting sequences can be used to restrict DNA nuclear import to specific cell types providing a new, novel means of cell targeting for gene therapy.

Cell-specific nuclear import of plasmid DNA in smooth muscle requires tissue-specific transcription factors and DNA sequences

Two shortcomings of nonviral gene therapy are a lack of tissue-specific targeting of vectors and low levels of gene transfer. Our laboratory has begun to address these limitations by designing plasmids that enter the nucleus of specific cell types in the absence of cell division, thereby enhancing expression in a controlled manner. We have shown that a 176 bp portion of the smooth muscle gamma-actin (SMGA) promoter can mediate plasmid nuclear import specifically in smooth muscle cells (SMCs). Here, we demonstrate that the binding sites for serum response factor (SRF) and NKX3-1/3-2 within this DNA nuclear targeting sequence (DTS) are required for plasmid nuclear import. Knockdown of these factors with siRNA abrogates plasmid nuclear import, indicating that they are necessary cofactors. In addition, coinjection of recombinant SRF and Nkx3.2 with the vector in TC7 epithelial cells rescues import. Finally, we show that the SRF nuclear localization sequence (NLS) is required for vector nuclear import. We propose that SRF and NKX3-1/3-2 bind the SMGA DTS in the cytoplasm, thus coating the plasmid with NLSs that mediate translocation across the nuclear pore complex. This discovery could aid in the development of more efficient nonviral vectors for gene transfer to SMCs.

329. Electroporation-Mediated Delivery of Cre- Expressing Plasmids to the Lung Results in Tissue-Specific DNA Excision in Transgenic Mice

Mouse models of human disease are an indispensable tool in elucidating pathology at the molecular level. Transgenic and knockout technologies are commonly used to establish the function of specific genes in both normal and pathological states. Recently, the advent of the Cre/loxP system of site-directed DNA recombination has taken such ‘reverse genetic’ methodologies a step further, allowing for temporal and spatial regulation of gene excision. This added level of control has permitted analysis of knockouts that would otherwise be embryonic lethal. However, despite the progress of the field, all of these techniques are incredibly time-consuming and cost-intensive. In this study, we describe the utility of plasmid DNA technology in inducing tissue-specific and temporally regulated gene excision that is highly efficient, rapid and economical. Previously, we have demonstrated that plasmid constructs containing certain DNA sequences and promoters can achieve nuclear entry and transcription of their encoded transgenes both in vitro and in vivo. Constructs lacking these nuclear import sequences, which we term DNA nuclear targeting sequences (DTSs), remain confined to the cytoplasm until cell division or until they are degraded. Many of the identified DTSs are from mammalian promoters. Due to the fact that promoters can drive transcription in a tissue-specific manner, plasmids containing these sequences can limit nuclear import and transgene expression to particular tissues. Here, we show that this targeting strategy can be used to express Cre-recombinase in a tissue-specific manner to create DNA deletions in the lungs of the Rosa reporter strain of transgenic mice. Rosa transgenic mice contain a lacZ reporter gene that is transcriptionally silenced by a floxed stop sequence immediately upstream. Upon expression of Cre-recombinase in target tissues, the stop sequence is cleaved, and the beta-galactosidase reporter is transcribed. To achieve this tissue-specific excision of the DNA sequence, we constructed plasmids that contain: (1) the Cre- recombinase expression cassette and (2) promoter sequences (DTSs) that mediate nuclear import (and hence expression) specifically in the lung. These plasmids were then placed in a saline suspension and electroporated into the lungs of anesthetized Rosa mice. Using this method, we have shown that the mammalian CMV promoter can successfully direct Cre-mediated DNA excision in the lung, as demonstrated by beta-galactosidase expression. We are currently testing the smooth muscle gamma-actin (SMGA) promoter for smooth muscle-specific excision. This ability to achieve temporally and spatially regulated gene excision with plasmid constructs offers an enticing alternative to complex breeding strategies that are normally employed in Cre/loxP studies.

Urogenital Tract Expression of Enhanced Green Fluorescent Protein in Transgenic Mice Driven by a Smooth Muscle γ-Actin Promoter

Our understanding of urogenital tract development and its response to disease or injury is hindered by complex interactions between epithelial and mesenchymal cells, and the difficulties in studying either component in isolation. We investigated whether transgenic mice could be generated to express enhanced green fluorescent protein (EGFP) in smooth muscle cells (SMCs) and whether such cells could then be purified using flow cytometric sorting to isolate RNA to be used in future gene expression assays. A 13.7 kb mouse smooth muscle gamma-actin promoter fragment was ligated to an EGFP reporter gene and microinjected into male mouse pronuclei. Adult transgenic mice were sacrificed and urogenital tissues were removed for histological and immunohistochemical studies. In other animals conditions were determined for dissociating bladder cells and the subsequent purification of bladder SMCs by sorting. Six lines of transgenic mice were generated (transgene copy numbers 1 to 30). EGFP was expressed in all smooth muscle beds examined except those associated with small blood vessels. EGFP levels appeared to correlate with transgene copy number. Histological and immunohistochemical analysis confirmed that reporter gene expression was restricted to SMCs of all tissues examined. Parameters for generating bladder cell suspensions were established and EGFP labeled bladder SMCs were identified by flow cytometric analysis. Several lines of transgenic mice have been generated in which SMCs of urogenital tissues have been labeled with EGFP and pure populations of SMCs have been obtained. The methods established for the rapid dissociation and purification of bladder SMCs should minimize degradative changes. These approaches may enable us to address issues involving bladder SMC development and differentiation as well as the response to injury and disease by performing transcriptome wide analyses on purified SMC populations.

The smooth muscle gamma-actin gene is androgen responsive in prostate epithelia.

Nkx 3.1 is an evolutionarily conserved vertebrate homolog of the Drosophila Nk-3 homeodomain gene bagpipe that is expressed by a variety of cells during early mammalian development and has been shown to be a critical factor for prostate development and function. Previous studies utilizing a heterologous cell transfection strategy from our laboratory identified the smooth muscle gamma-actin (SMGA) gene as a novel molecular target of Nkx 3.1 regulatory activity. In the studies presented here, SMGA gene activity and regulation were evaluated in normal and cancerous prostate epithelial cells. SMGA transcripts were demonstrated in prostate epithelia and SMGA mRNA levels were increased in androgen-responsive LNCaP cancer and normal prostate epithelial cells. SMGA gene transcriptional activity was androgen responsive in these cells and required a segment of the human SMGA promoter containing NKE and SRF (serum response factor) binding elements. This region of the human SMGA proximal promoter is well conserved across species and is synergistically activated by coexpression of Nkx 3.1 and SRF in heterologous CV-1 cells. SMGA transcription was not responsive to steroid in PC-3 prostate epithelial cancer cells, which do not express Nkx 3.1. However, SMGA transcription was influenced by expression of androgen receptor in these cells, a situation that allows the androgen-dependent expression of Nkx 3.1. Furthermore, SMGA gene activity was influenced by direct Nkx 3.1 expression in the PC-3 cells. Thus, SMGA gene activity in prostate epithelia is due, in part, to the androgen-dependent expression of Nkx 3.1. As such, our studies provide the initial description of Nkx 3.1 target gene regulatory activity in the prostate.

The Smooth Muscle γ-Actin Gene Promoter Is a Molecular Target for the Mouse bagpipe Homologue, mNkx3-1, and Serum Response Factor

An evolutionarily conserved vertebrate homologue of the Drosophila NK-3 homeodomain gene bagpipe, Nkx3-1, is expressed in vascular and visceral mesoderm-derived muscle tissues and may influence smooth muscle cell differentiation. Nkx3-1 was evaluated for mediating smooth muscle gamma-actin (SMGA) gene activity, a specific marker of smooth muscle differentiation. Expression of mNkx3-1 in heterologous CV-1 fibroblasts was unable to elicit SMGA promoter activity but required the coexpression of serum response factor (SRF) to activate robust SMGA transcription. A novel complex element containing a juxtaposed Nkx-binding site (NKE) and an SRF-binding element (SRE) in the proximal promoter region was found to be necessary for the Nkx3-1/SRF coactivation of SMGA transcription. Furthermore, Nkx3-1 and SRF associate through protein-protein interactions and the homeodomain region of Nkx3-1 facilitated SRF binding to the complex NKE.SRE. Mutagenesis of Nkx3-1 revealed an inhibitory domain within its C-terminal segment. In addition, mNkx3-1/SRF cooperative activity required an intact Nkx3-1 homeodomain along with the MADS box of SRF, which contains DNA binding and dimerization structural domains, and the contiguous C-terminal SRF activation domain. Thus, SMGA is a novel target for Nkx3-1, and the activity of Nkx3-1 on the SMGA promoter is dependent upon SRF.

Ultrastructural and histochemical characterization of special muscle cells in the monkey small intestine.

The ultrastructure, three-dimensional arrangement, and histochemical features of special muscle cells in the monkey small intestine were investigated. The cells formed a special layer separated from the main part by a connective tissue space along the submucosal surface of the circular muscle coat. Scanning electron microscopy using alkali maceration demonstrated this inner sublayer to be a continuous thin sheet consisting of irregularly-shaped muscle cells equipped with many cytoplasmic projections and caveolae. Other ultrastructural features included direct contact with interstitial cells, due to their close association with nerve fibers of the deep muscular plexus. Histochemical examination revealed significant alkaline phosphatase activity and immunoreactivity for vascular smooth muscle alpha actin in these muscle cells, whereas the ordinary circular muscle cells were immunopositive for enteric smooth muscle gamma actin. These findings suggest that the special muscle cells play an important role in regulating the radial stretch of the monkey small intestinal wall.

Cell-specific nuclear import of plasmid DNA.

One factor limiting the success of non-viral gene therapy vectors is the relative inability to target genes specifically to a desired cell type. To address this limitation, we have begun to develop cell-specific vectors whose specificity is at the level of the nuclear import of the plasmid DNA. We have recently shown that nuclear import of plasmid DNA is a sequence-specific event, requiring the SV40 enhancer, a region known to bind to a number of general transcription factors (Dean DA, Exp Cell Res 1997; 230: 293). From these studies we developed a model whereby transcription factor(s) bind to the DNA in the cytoplasm to create a protein-DNA complex that can enter the nucleus using the protein import machinery. Our model predicts that by using DNA elements containing binding sites for transcription factors expressed in unique cell types, we should be able to create plasmids that target to the nucleus in a cell-specific manner. Using the promoter from the smooth muscle gamma actin (SMGA) gene whose expression is limited to smooth muscle cells, we have created a series of reporter plasmids that are expressed selectively in smooth muscle cells. Moreover, when injected into the cytoplasm, plasmids containing portions of the SMGA promoter localize to the nucleus of smooth muscle cells, but remain cytoplasmic in fibroblasts and CV1 cells. In contrast, a similar plasmid carrying the SV40 enhancer is transported into the nuclei of all cell types tested. Nuclear import of the SMGA promoter-containing plasmids could be achieved when the smooth muscle specific transcription factor SRF was expressed in stably transfected CV1 cells, supporting our model for the nuclear import of plasmids. Finally, these nuclear targeting sequences were also able to promote increased gene expression in liposome- and polycation-transfected non-dividing cells in a cell-specific manner, similar to their nuclear import activity. These results provide proof of principle for the development of cell-specific non-viral vectors for any desired cell type.

ALTERATIONS IN INTESTINAL SMOOTH MUSCLE IN THE ABSENCE OF PACEMAKER ACTIVITY

Interstitial Cells of Cajal [ICC] generate pacemaker slow waves in the murine gastrointestinal [GI] tract which conduct through the gap junctions to the smooth muscle cells. ICC express the proto-oncogene c-kit. W/Wv mice have a mutation of c-kit and show a marked reduction in the number of ICC and an absence of pacemaker waves. To test the hypothesis that loss of ICC alters the smooth muscle phenotype, we compared the GI tract in W/Wv mice with that of age and sex matched controls. Smooth muscle alpha actin [SMAA] and smooth muscle gamma actin [SMGA] were quantified and compared in the fundus, body, pylorus, duodenum, jejunum, ileum, proximal and distal colon. In both the mutant and control animals at 8 weeks of age, the levels of these proteins varied in a segment specific pattern with pylorus, doudenum and jejunum showing a much lower expression of both actins relative to the other segments. However, W/Wv mice demonstrated decreased levels of SMGA and SMAA throughout the GI tract. The mutants also displayed an enlarged proximal gut but no changes were observed in smooth muscle histology. At 4 weeks of age, no differences were noted by these criteria. This study represents the first comprehensive analysis of smooth muscle isoactin expression at the protein level in the adult murine GI tract. The results show that smooth muscle isoactin content is segment-specific and that the level of these proteins is reduced in the absence of pacemaker activity. Although the basis for the late onset of these changes and the enlargement of the stomach and duodenum remains to be determined, this may reflect pseudo-obstruction of the gut and W/Wv mice could provide an animal model for this disorder.

Establishment and characterization of a conditionally immortalized smooth muscle/myometrial-like cell line.

A novel smooth muscle/myometrial-like cell line, SMU1-10, has been generated from the uterus of a H-2Kb-tsA58 transgenic mouse carrying a thermolabile SV40 large T-antigen gene. These cells grow continuously when maintained at the permissive temperature (33 degrees C) for the SV40 large T-antigen but stop dividing when placed at the non-permissive temperature (39 degrees C) and ultimately die within 3 weeks. All of the SMU1-10 cells produce smooth muscle alpha-actin (SMAA) at both 33 degrees C and 39 degrees C. A subset of the cells also contain smooth muscle gamma-actin (SMGA), a hallmark of smooth muscle differentiation, and the fraction of cells staining for this actin increases from about 1% when maintained for three days at 33 degrees C to as much as 30% at 39 degrees C over the same length of time. However, the appearance of SMGA in SMU1-10 cells appears to be regulated mainly at a post-transcriptional level since in situ hybridization indicates that all cells contain SMGA mRNA at both 33 degrees C and 39 degrees C. SMU1-10 cultures also contain smooth muscle myosin heavy chain (SM-MHC) and SM22 alpha, both of which are only found in smooth muscle of the adult mouse. Three additional smooth muscle (myometrium)-related markers, connexin 43, the thromboxane A2 receptor, and the progesterone receptor also are present in these cells. At the nonpermissive temperature for SV40 large T-antigen, the both level of SMGA mRNA and the number of cells staining for this actin are significantly increased in the presence of progesterone, a process that is similar to the upregulation of SMGA in the myometrium late in pregnancy. Overall, SMU1-10 cells provides a potentially useful in vitro model system to study smooth muscle/myometrial differentiation.

Tissue and developmental specific expression of murine smooth muscle gamma-actin fusion genes in transgenic mice.

Smooth muscle gamma-actin (SMGA) is an excellent marker of smooth muscle differentiation because it is essentially restricted to smooth muscle. As a first step toward unraveling the mechanisms underlying smooth muscle development and differentiation, we have examined the tissue-specific and developmental expression patterns of six constructs carrying portions of the murine SMGA gene linked to chloramphenicol acetyltransferase (CAT) in stable lines of transgenic mice. Based on the transgenic studies most, if not all, of the regulatory elements necessary for proper spatial and temporal expression of SMGA are present within a 13.7 kb segment of the SMGA gene containing 4.9 kb of upstream sequence, exon 1, intron 1, and a portion of exon 2 up to the start codon for translation. A second construct (SMGA11.6CAT) that lacks the distal 2.1 kb of upstream sequence but is otherwise identical to SMGA13.7CAT shows a similar level of smooth muscle-specific CAT activity. However, SMGA9.3CAT fusion gene containing only 571 bp of 5' flanking sequence, but otherwise identical to SMGA13.7CAT, and SMGA6.0CAT containing only the 4.9 kb upstream sequence, exon 1, and a miniintron 1 show a more than a 100-fold reduction of CAT activity in most smooth muscle-rich tissues. Furthermore, removal of most or all of intron 1 from a transgene with 571 bp of upstream sequence (SMGA2.0 CAT and SMGA0.6CAT) results in a near-complete or complete loss of activity, respectively, in all tissues. Overall, the studies suggest that upstream elements between -2.7 kb and -571 bp and elements within intron 1 are required for high levels of SMGA gene expression in an appropriate temporal-spatial fashion.

Identification and classification of interstitial cells in the canine proximal colon by ultrastructure and immunocytochemistry

The ultrastructure and immunocytochemistry of interstitial cells (ICs) in the canine proximal colon were investigated. Three types of ICs were found within the tunica muscularis. (1) ICs were located along the submucosal surface of the circular muscle (IC-SM). These cells shared many features of smooth muscle cells, including myosin thick filaments and immunoreactivity to smooth muscle gamma actin, myosin light chain, and calponin antibodies. IC-SM were clearly different from smooth muscle cells in that contractile filaments were less abundant and intermediate filaments consisted of vimentin instead of desmin. (2) ICs in the region of the myenteric plexus (IC-MY) were similar to IC-SM, but these cells had no thick filaments or immunoreactivity to smooth muscle gamma actin or calponin antibodies. (3) The fine structures and immunoreactivity of ICs within the muscle layers (IC-BU) were similar to IC-MY, but IC-BU lacked a definite basal lamina and membrane caveolae. IC-BU and IC-MY were both immunopositive for vimentin. Since all ICs were immunopositive for vimentin, vimentin antibodies may be a useful tool for distinguishing between ICs and smooth muscle cells. Each class of ICs was closely associated with nerve fibers, made specialized contacts with smooth muscle cells, and formed multicellular networks. A combination of ultrastructural and immunocytochemical techniques helps the identification and classification of ICs by revealing the fine structures and determining the "chemical coding" of each class of ICs.

Alterations in the expression of the beta-cytoplasmic and the gamma-smooth muscle actins in hypertrophied urinary bladder smooth muscle.

The obstruction of the bladder outlet induces a marked increase in bladder mass, and this is accompanied by reduced contractility of bladder smooth muscle and alteration in the cellular architecture. In this study, we show that the composition of various isoforms of actin, a major component of the contractile apparatus and the cytoskeletal structure of smooth muscle, is altered in response to the obstruction-induced bladder hypertrophy. Northern blot analysis of the total RNA isolated from hypertrophied urinary bladder muscle, using a cDNA probe specific for smooth muscle gamma-actin, shows over 200% increase in the gamma-actin mRNA. However, the estimate of the amount of actin from the 2D gel reveals only a 16% increase in gamma-actin, since the 2D gel electrophoresis does not distinguish gamma-smooth muscle actin from gamma-cytoplasmic actin. The bladder smooth muscle alpha-actin and the smooth muscle alpha-actin mRNA are not altered in response to the hypertrophy. The obstructed bladder also reveals a decrease in the beta-cytoplasmic actin (37%) and a concomitant diminution in the beta-cytoplasmic actin mRNA (29%). Hence, the composition of the actin isoforms in bladder smooth muscle is altered in response to the obstruction-induced hypertrophy. This alteration of the actin isoforms is observed at both the protein and mRNA levels.

Molecular cloning and expression of the chicken smooth muscle gamma-actin mRNA.

We have investigated the expression of chicken smooth muscle gamma-actin mRNA by isolation and characterization of cDNAs representing this actin isoform and utilizing the cDNA to probe RNA from adult and developing cells. Nucleotide sequence elucidated from an apparent full length smooth muscle gamma-actin cDNA revealed that it contained 94 bp of 5' non-translated sequence, an open reading frame of 1131 bp, and 97 bp of 3' non-translated sequence. Within the 376 amino acid sequence deduced from the chicken cDNA were diagnostic amino acids at the NH2- and COOH-terminal regions which provided unequivocal identification of the gamma-enteric smooth muscle actin isoform. In addition, the chicken gamma-enteric actin deduced from our cDNA clones was found to differ from the sequence reported in earlier protein studies [J. Vandekerckhove and K. Weber, FEBS Lett. 102:219, 1979] by containing a proline rather than a glutamine at position 359 of the protein, indicating that the avian gamma-enteric actin isoform is identical to its mammalian counterpart. Comparison of the 5' and 3' non-translated sequence determined from the chicken cDNA to that elucidated for rat, mouse, and human showed that there is not a high degree of cross-species sequence conservation outside of the coding regions among these mRNAs. Northern hybridization analyses demonstrated that the gamma-enteric actin mRNA is expressed in adult aorta and oviduct tissues but not in adult skeletal muscle, cardiac muscle, liver, brain, and spleen tissues. The gamma-enteric actin mRNA was first observed in measurable quantities in gizzard tissue from 4-5 day embryos and increased in content in developing smooth muscle cells through 16-17 embryonic days. Following this initial increase during embryonic development, the gamma-enteric actin mRNA exhibits a decline in content until approximately 7 days posthatching, after which there is an increase in content to maximal levels found in adult gizzard tissue. In general, the developmental appearance of the gamma-enteric mRNA parallels that observed for this protein in previous studies indicating that the developmental expression of smooth muscle gamma-actin is regulated, in part, by an increased content of mRNA in chicken visceral smooth muscle cells during myogenesis.

ADP‐ribosylation of actins by arginine‐specific ADP‐ribosyltransferase purified from chicken heterophils

We reported the purification and characterization of an arginine-specific ADP-ribosyltransferase and acceptor protein p33 in granules of chicken peripheral polymorphonuclear leukocytes (heterophils) [Mishima, K., Terashima, M., Obara, S., Yamada, K., Imai, K. & Shimoyama, M. (1991) J. Biochem. (Tokyo) 110, 388-394]. In the present study, we obtained evidence that chicken non-muscle beta/gamma-actin, skeletal muscle alpha-actin and smooth-muscle gamma-actin were ADP ribosylated by the heterophil ADP-ribosyltransferase. The stoichiometry of ADP-ribose incorporation into these actins was 1.2 mol, 1.0 mol and 2.0 mol ADP-ribose/mol of beta/gamma-actin, alpha-actin and gamma-actin, respectively. The optimal pH for the ADP ribosylation was at pH 8.5, with the respective actin. Km values for NAD were calculated to be 30 microM with beta/gamma-actin, 35 microM with alpha-actin and 20 microM with gamma-actin. The Km values for the actin isoforms were 15 microM for beta/gamma-actin, 2.5 microM for alpha-actin and 10 microM for gamma-actin. ADP ribosylation of actin inhibited its capacity to polymerize, as determined by the increase in fluorescence intensity with N-(1-pyrenyl)iodoacetamide-labelled actin. Filamentous actin (F-actin) polymerized with the respective actin isoform was also ADP ribosylated, although the extent of the modification of F-actin was lower than that of globular actin (G-actin). In situ ADP ribosylation of beta/gamma-actin was evidenced with chicken peripheral heterophils permeabilized with saponin. Thus, the endogenous ADP ribosylation of actin in the heterophils may be involved in the cellular processes such as phagocytosis, secretion and migration.

Structure, chromosome location, and expression of the human smooth muscle (enteric type) gamma-actin gene: evolution of six human actin genes.

Recombinant phages that carry the human smooth muscle (enteric type) gamma-actin gene were isolated from human genomic DNA libraries. The amino acid sequence deduced from the nucleotide sequence matches those of cDNAs but differs from the protein sequence previously reported at one amino acid position, codon 359. The gene containing one 5' untranslated exon and eight coding exons extends for 27 kb on human chromosome 2. The intron between codons 84 and 85 (site 3) is unique to the two smooth muscle actin genes. In the 5' flanking region, there are several CArG boxes and E boxes, which are regulatory elements in some muscle-specific genes. Hybridization with the 3' untranslated region, which is specific for the human smooth muscle gamma-actin gene, suggests the single gene in the human genome and specific expressions in enteric and aortic tissues. From characterized molecular structures of the six human actin isoform genes, we propose a hypothesis of evolutionary pathway of the actin gene family. A presumed ancestral actin gene had introns at least sites 1, 2, and 4 through 8. Cytoplasmic actin genes may have directly evolved from it through loss of introns at sites 5 and 6. However, through duplication of the ancestral actin gene with substitutions of many amino acids, a prototype of muscle actin genes had been created. Subsequently, striated muscle actin and smooth muscle actin genes may have evolved from this prototype by loss of an intron at site 4 and acquisition of a new intron at site 3, respectively.