Differential binding of an SRF/NK-2/MEF2 transcription factor complex in normal versus neoplastic smooth muscle tissues.

The malignant potential of smooth muscle tumors correlates strongly with the disappearance of gamma-smooth muscle isoactin, a lineage-specific marker of smooth muscle development. In this paper, we identify a 36-base pair regulatory motif containing an AT-rich domain, CArG box, and a non-canonical NK-2 homeodomain-binding site that has the capacity to regulate smooth muscle-specific gene expression in cultured intestinal smooth muscle cells. Serum-response factor associates with an NK-2 transcription factor via protein-protein interactions and binds to the core CArG box element. Our studies suggest that the NK-2 transcription factor that associates with serum-response factor during smooth muscle differentiation is Nkx2-3. Myocyte-specific enhancer factor 2 binding to this regulatory complex was also observed but limited to uterine smooth muscle tissues. Smooth muscle neoplasms displayed altered transcription factor binding when compared with normal myometrium. Differential nuclear accessibility of serum-response factor protein during smooth muscle differentiation and neoplastic transformation was also observed. Thus, we have identified a unique regulatory complex whose differential binding properties and nuclear accessibility are associated with modulating gamma-smooth muscle isoactin-specific gene expression in both normal and neoplastic tissues.

Characteristics of inflammation-induced hypertrophy of rat intestinal smooth muscle cell.

Inflammation of the human intestine causes thickening of the smooth muscle layers, and studies in rats infected with Trichinella spiralis (Tsp) have shown hyperplasia of the intestinal smooth muscle cells (ISMC). We have shown that Tsp-induced inflammation caused a fivefold increase in total protein per ISMC over control, while ISMC from the noninflamed distal ileum also showed a threefold increase. The amount of alpha-smooth muscle (SM) actin per ISMC increased nearly 500% over control by postinfection (PI) day 6. The proportion of alpha-SM actin in the total cellular protein increased 200% by day 6 PI, indicating a higher density of alpha-SM actin in the hypertrophied ISMC. Gamma-SM actin mRNA increased sharply and was matched by an increased fractional content of gamma-SM actin protein. These increases in the smooth muscle-specific actins may affect force production and further demonstrate the plasticity of smooth muscle in the inflamed intestine.

Differential display identification of plunc, a novel gene expressed in embryonic palate, nasal epithelium, and adult lung.

We have identified a novel gene transcript of approximately 1.1 kilobases in length that is expressed in the presumptive nasal epithelium of the mouse embryo. In situ hybridization analysis shows discrete regions of expression associated with the palate, nasal septum, and nasal conchae. This transcript is also expressed strongly in the trachea and bronchi of the adult lung. Screening of a mouse heart cDNA library yielded several overlapping clones to give a continuous sequence of 1113 bases, containing an open reading frame of 278 codons comprising the complete mRNA. No significant homologies with known genes were observed at the nucleotide level; limited amino acid homology with two salivary gland-specific proteins was noted. A search for functionally significant protein motifs revealed consensus sequences for N-glycosylation, protein kinase C and casein kinase phosphorylation, and a leucine zipper. Additionally, we observed a unique amino acid sequence pattern, consisting of the residues Gly-(Leu/Pro/Gln)-(Pro/Leu)-Leu-Pro-Leu, repeated four times near the amino-terminal portion of the protein with two amino acid residues separating the repeats. Based on these observations, we propose that we have identified a new gene, which we call plunc (for palate, lung, and nasal epithelium clone; GenBankTM accession number U69172).

Identification of distinct molecular phenotypes in cultured gastrointestinal smooth muscle cells.

BACKGROUND & AIMS: Cultured gastrointestinal smooth muscle cells have been shown to dedifferentiate and reinitiate their myogenic program in vitro. The aim of this study was to determine whether the cellular phenotypes observed in vitro were similar to those previously characterized in vivo. METHODS: Differential isoactin expression was examined in primary cultures of intestinal smooth muscle cells (ISMCs) by Northern blot and immunohistochemical analysis. Cellular phenotype was determined for cultured ISMCs grown at high density, at low density, in the presence and absence of serum supplementation, and on several distinct substrates including collagen type IV, laminin, fibronectin, and plastic. RESULTS: The unique patterns of isoactin protein and gene expression observed in cultured ISMCs indicate that distinct cellular phenotypes were present in vitro. The production and maintenance of these distinct smooth muscle cell phenotypes was dependent on cell density, serum supplementation, and substrate used. CONCLUSIONS: Cultured ISMCs appear to recapitulate a portion of their in vivo myogenic program in vitro, providing a unique opportunity for the molecular mechanisms controlling gastrointestinal smooth muscle myogenesis and pathogenesis to begin to be identified.

Smooth muscle actin and myosin expression in cultured airway smooth muscle cells.

In this study, the expression of smooth muscle actin and myosin was examined in cultures of rat tracheal smooth muscle cells. Protein and mRNA analyses demonstrated that these cells express alpha- and gamma-smooth muscle actin and smooth muscle myosin and nonmuscle myosin-B heavy chains. The expression of the smooth muscle specific actin and myosin isoforms was regulated in the same direction when growth conditions were changed. Thus, at confluency in 1 or 10% serum-containing medium as well as for low-density cells (50-60% confluent) deprived of serum, the expression of the smooth muscle forms of actin and myosin was relatively high. Conversely, in rapidly proliferating cultures at low density in 10% serum, smooth muscle contractile protein expression was low. The expression of nonmuscle myosin-B mRNA and protein was more stable and was upregulated only to a small degree in growing cells. Our results provide new insight into the molecular basis of differentiation and contractile function in airway smooth muscle cells.

Molecular determination of the malignant potential of smooth muscle neoplasms.

BACKGROUND: The determination of the malignant potential of smooth muscle neoplasms remains ambiguous, and yet has far reaching clinical, therapeutic, and social implications. METHODS: In this pilot study, the authors examined smooth muscle isoactin gene expression by polymerase chain reaction in a variety of smooth muscle tumors. RESULTS: A lack of gamma-smooth muscle isoactin gene expression correlated 100% with a pathologic diagnosis of sarcoma. These results suggest that gamma-smooth muscle isoactin gene expression represents a unique molecular marker of oncogenic transformation. CONCLUSIONS: gamma-Smooth muscle isoactin gene expression provides a valuable molecular adjunct to the diagnosis of smooth muscle neoplasms.

Inhibitory neurotransmission in lethal spotted mutant mice: a model for Hirschsprung's disease.

BACKGROUND & AIMS: The pathogenesis of Hirschsprung's disease is not well understood. The suitability of the animal model for the unknown pathogenesis of inhibitory neurotransmission in Hirschsprung's disease was investigated. METHODS: Circular smooth muscle strips from the internal anal sphincter (IAS) and distal colon (2, 6, 8, 16, and 24 mm from the anal verge) from normal and Ls/Ls mice (mice homozygous for the lethal spotting mutation that develop fetal megacolon after aganglionosis of the terminal colon) were prepared to record changes in isometric tensions in response to different agents and nonadrenergic, noncholinergic nerve stimulation by electrical field stimulation. RESULTS: Bethanechol was used to produce contraction of the smooth muscle strips of distal colon to record a decrease in the tension. Conversely, the IAS smooth muscle strips developed spontaneous tone. In the normal homozygous mice, electrical field stimulation caused a biphasic response, an initial decrease followed by an after-contraction, whereas in Ls/Ls mice, the predominant response was contraction. All smooth muscle strips from normal and Ls/Ls mice produced relaxation in response to sodium nitroprusside and vasoactive intestinal polypeptide. CONCLUSIONS: Ls/Ls mice may serve as an appropriate animal model to investigate the pathogenesis of the inhibitory neurotransmission in Hirschsprung's disease in the distal colon and IAS.

Comparative analysis of smooth muscle isoactin gene expression in normal and neoplastic tissues.

Monoclonal antibodies derived from the actin multigene family are routinely used as an adjunct to morphologic diagnoses of smooth muscle tumors. Northern blot analysis was performed on 60 surgical resections utilizing isoactin-specific cDNAs. A comparison of this analysis to immunohistochemical studies demonstrated that actin-specific monoclonal antibodies represent reliable markers of the smooth muscle lineage. Smooth muscle neoplasms showed a unique pattern of gamma-smooth muscle isoactin gene expression, providing a potentially valuable molecular adjunct to the morphologic diagnosis of uterine smooth muscle tumors.

Isolation and characterization of a novel short chain alcohol dehydrogenase-like isozyme by differential display of distinct smooth muscle cell phenotypes.

Gastrointestinal smooth muscle development proceeds by the linear differentiation of distinct smooth muscle cell phenotypes. In an effort to identify specific gene products associated with distinct smooth muscle cell phenotypes, we performed differential display on smooth muscle myoblasts versus immature smooth muscle myocytes. This analysis identified a novel short-chain alcohol dehydrogenase-like isozyme which was preferentially expressed in smooth muscle myoblasts over immature and mature smooth muscle myocytes. We postulate that this novel short-chain alcohol dehydrogenase-like isozyme may play a role in potentiating the dedifferentiation of smooth muscle cells in vitro.

Molecular analysis of smooth muscle development in the mouse.

Little is currently known regarding the ontogeny of smooth muscle tissues during normal mammalian development. The alpha-smooth muscle and gamma-smooth muscle isoactins have been shown to be excellent molecular markers of smooth muscle cell phenotype. This study characterizes both the temporal and spatial patterns of alpha-smooth muscle and gamma-smooth muscle isoactin expression in the developing mouse. In situ analysis was performed on serial sections of whole mouse embryos on embryonic day 9, 11, 13, 15, and 17 using alpha-smooth muscle and gamma-smooth muscle isoactin-specific riboprobes. Distinct temporal and spatial patterns of alpha-smooth muscle and gamma-smooth muscle isoactin gene expression were observed in the developing gastrointestinal tract, urogenital tract, respiratory tract, and vascular system. Independent expression of the alpha-smooth muscle isoactin was observed during the early stages of skeletal, cardiac, and smooth muscle myogenesis as well as in a novel subset of distinct organs including the postnatal component of the hindgut, allantois, and primitive placenta. The results of this study indicate that distinct cellular phenotypes are involved in smooth muscle myogenesis and suggest that organ-specific mechanisms might exist for the initiation of smooth muscle development in vivo. In addition, the pattern of independent alpha-smooth muscle isoactin expression observed in this study provides novel information regarding the early stages of hindgut and placental development, and suggests that a common functional phenotype may be associated with the early stages of skeletal, cardiac, and smooth muscle myogenesis.

A high-resolution linkage map of the lethal spotting locus: a mouse model for Hirschsprung disease.

Mice homozygous for the lethal spotting (ls) mutation exhibit aganglionic megacolon and a white spotted coat owing to a lack of neural crest-derived enteric ganglia and melanocytes. The ls mutation disrupts the migration, differentiation, or survival of these neural crest lineages during mammalian development. A human congenital disorder, Hirschsprung disease (HSCR), is also characterized by aganglionic megacolon of the distal bowel and can be accompanied by hypopigmentation of the skin. HSCR has been attributed to multiple loci acting independently or in combination. The ls mouse serves as one animal model for HSCR, and the ls gene may represent one of the loci responsible for some cases of HSCR in humans. This study uses 753 N2 progeny from a combination of three intersubspecific backcrosses to define the molecular genetic linkage map of the ls region and to provide resources necessary for positional cloning. Similar to some cases of HSCR, the ls mutation acts semidominantly, its phenotypic effects dependent upon the presence of modifier genes segregating in the crosses. We have now localized the ls mutation to a 0.8-cM region between the D2Mit113 and D2Mit73/D2Mit174 loci. Three genes, endothelin-3 (Edn3), guanine nucleotide-binding protein alpha-stimulating polypeptide 1 (Gnas), and phosphoenolpyruvate carboxykinase (Pck1) were assessed as candidates for the ls mutation. Only Edn3 and Gnas did not recombine with the ls mutation. Mutational analysis of the Edn3 and Gnas genes will determine whether either gene is responsible for the neural crest deficiencies observed in ls/ls mice.

Differential isoactin gene expression in the sphincteric and nonsphincteric gastrointestinal smooth muscles of the opossum.

The sphincteric smooth muscle tissues of the gastrointestinal tract have been shown to possess mechanical properties distinct from the surrounding nonsphincteric smooth muscle tissues. Little is currently known regarding the molecular basis of this differential smooth muscle development and function. Actin is an important contractile protein whose expression has been linked to the normal development and function of smooth muscle tissues. The purpose of this study was to characterize isoactin gene expression in the sphincteric versus nonsphincteric smooth muscle tissues of the gastrointestinal tract. Northern blot analysis was performed on manometrically identified sphincteric and the flanking nonsphincteric smooth muscle tissues of the adult opossum. Quantitative analysis revealed a distinct pattern of isoactin gene expression in the sphincteric versus nonsphincteric smooth muscle tissues of the gut. The sphincteric smooth muscle tissues expressed significantly lower total quantities of isoactin mRNA than their surrounding nonsphincteric smooth muscle tissues. It is hypothesized that these differential patterns of isoactin gene expression may play a significant role in establishing the myogenic potential of the functionally distinct sphincteric and nonsphincteric smooth muscle tissues of the gut.

Heterogeneous isoactin gene expression in the adult rat gastrointestinal tract.

BACKGROUND: Normal gastrointestinal development is a complex process involving the precise integration of multiple cell types. To gain a better understanding of these processes, the present study examined isoactin gene expression in the adult rat gastrointestinal tract. METHODS: Northern blot analysis was performed on specified segments of the adult rat esophagus, stomach, small intestine, cecum, colon, rectum, and anus using actin isoform-specific complementary DNAs for all six vertebrate isoactins. RESULTS: Smooth muscle and cytoplasmic isoactins were heterogeneously coexpressed in a segment-specific manner throughout the gastrointestinal tract. In addition, striated muscle isoactin expression was also detected in segments of the adult rat esophagus, stomach, colon, cecum, rectum, and anus. Histological analysis indicated that the adult rat esophagus, stomach, and anus contained significant quantities of skeletal muscle, providing a source for the striated muscle isoactins detected in these gut segments. A similar source of striated muscle isoactin expression in the cecum, colon, and rectum was not identified. Both coordinate and independent regulation of isoactin gene expression was observed in the gastrointestinal tract, although distinct patterns of autoregulation were absent. CONCLUSIONS: This study represents the first complete analysis of isoactin gene expression in the adult rat gastrointestinal tract and provides the basis for future studies designed to investigate the factors responsible for these processes.

Altered isoactin gene expression in the affected bowel segments of the lethal spotted mouse.

BACKGROUND: Actin is a key contractile protein associated with the normal differentiation and function of gastrointestinal smooth muscle cells. Distinct changes in gastrointestinal smooth muscle cell morphology and function have been reported for the aganglionic rectum and megacolon of the adult lethal spotted mouse. This study examines what effect these changes in smooth muscle cell morphology and function have on the expression of the actin multigene family in both the aganglionic rectum and megacolon of the lethal spotted mouse. METHODS: Expression of the smooth muscle and cytoplasmic isoactins was examined by Northern blot analysis of the aganglionic rectum and megacolon of the homozygotic lethal spotted mouse and the equivalent bowel segments of control animals. RESULTS: The megacolon of the lethal spotted mouse showed a significant increase in gamma-smooth muscle isoactin expression. The aganglionic rectum of the lethal spotted mouse displayed a complex pattern of altered isoactin gene expression that included changes in both gamma-smooth muscle and beta-cytoplasmic isoactin expression. Strain-specific differences in the quantitative levels of isoactin gene expression were observed for the various bowel segments examined in this study. CONCLUSIONS: These results show that the changes in smooth muscle cell morphology and function observed in the lethal spotted mutant mouse are accompanied by significant alterations in isoactin gene expression.

A comprehensive analysis of the developmental and tissue-specific expression of the isoactin multigene family in the rat.

The present study represents the first comprehensive analysis of isoactin gene expression in the developing rat. Our results clearly demonstrate that the developmental and tissue-specific expression of the actin multigene family is a highly integrated and complex process involving a variety of regulatory paradigms. The distinct temporal patterns of expression reported in this study indicate that there are three key phases in the regulation of expression of the actin multigene family during development. These include early embryonic development, late fetal development, and early postnatal development. The specific spatial patterns of expression observed in this study demonstrate that the expression of the actin multigene family is much more permissive than previously reported. This permissive expression includes a wide range of "ectopic" expression of the striated muscle isoactins as well as an extended expression of the alpha-smooth muscle isoactin. These findings expand our current understanding of the expression of the actin multigene family in development and provide a fundamental basis for future studies directed at investigating these processes.

Persistent karyomegaly caused by Penicillium nephrotoxins in the rat.

Continuous or intermittent consumption by rats of food moulded by Penicillium aurantiogriseum induced prominent and extensive histopathological changes within several weeks seen specifically at the renal cortico-medullary junction. Many cells of the P3 segment of proximal tubules contained either giant nuclei or multiple enlarged nuclei, described in this text as karyomegaly, but also included within a cytomegalic change. The changes contrasted with the tubular cell necrosis and concomitant mitosis elicited after only four days consumption of nephrotoxic mould. Unilateral nephrectomy enabled persistence of histopathological changes to be assessed directly after detailed histology at an earlier stage. After ten days consumption of food with a 100-fold excess of fungal extract containing the amphoteric nephrotoxins, the typical acute histopathology evolved, over a period of three weeks on normal diet, into the bizarre karyomegalic histopathology, implying a latent effect. Karyomegaly persisted for at least twelve months after nephrotoxin dosage ceased. P. aurantiogriseum karyomegaly was much more striking than that induced by a relatively high chronic dose of another Penicillium nephrotoxin, ochratoxin A. Although the study does not attempt to measure relative potencies, qualitatively similar ultrastructural changes (enlarged nuclei, proliferation of smooth endoplasmic reticulum and thickening of proximal tubule basement membranes) were induced by the two types of nephrotoxin. The broadly toxic ochratoxin A is the popular putative aetiological agent in the mysterious and insidious Balkan endemic nephropathy and associated urinary tract tumours. As the renal carcinogenicity of ochratoxin A in rats follows karyomegaly, the striking karyomegaly induced by P. aurantiogriseum in the proximal tubules of the kidney must be considered as a potential factor in human chronic renal disease.

Penicillium aurantiogriseum-induced, persistent renal histopathological changes in rats; an experimental model for Balkan endemic nephropathy competitive with ochratoxin A.

Renal histopathological changes in rats, caused by food partially moulded by a common fungus isolated from an area of nephropathy in Yugoslavia, were differentiated into acute and chronic responses. The acute response to a few days on the diet specifically involved necrosis and concomitant mitosis in proximal tubule cells. More protracted, continuous or intermittent administration of nephrotoxic mould led to a marked karyomegaly in the same corticomedullary region. The phenomenon is more prominent than that induced by treatment with ochratoxin A over a similar period, raising the question of the putative role of such mycotoxins in the etiology of chronic human renal disease.

Insulin-mediated regulation of epididymal fat pad malic enzyme.

The complex nature of insulin-mediated biological responses has made it difficult to interpret such data. Prior studies in our laboratory had characterized the insulin-mediated increases in hepatic malic enzyme activity in normal and diabetic rats (Drake et al., 1983; Drake and Mucenski, 1985). However, since insulin-mediated regulatory processes have been shown to be tissue specific, we decided to examine malic enzyme activity in the epididymal fat pads of normal, diabetic, and insulin-treated normal and diabetic rats. This data revealed that in direct contrast to the hepatic studies, the normal epididymal fat pad contained the low specific activity malic enzyme molecule. Insulin treatment of both normal and diabetic rats resulted in an increase in epididymal fat pad malic enzyme activity due to increases in both enzyme quantity and specific activity. In order to quantitate epididymal fat pad malic enzyme mRNA levels, we isolated a 2.4 kb malic enzyme specific cDNA which was designated pR ME 1. In both normal and diabetic rats, the observed increases in malic enzyme quantity were directly paralleled by increases in malic enzyme mRNA content. The Northern blot data revealed an apparent differential expression of the malic enzyme mRNA doublet between insulin-treated normal and diabetic epididymal fat pads. This study demonstrates that insulin modulates epididymal fat pad and hepatic malic enzyme activity in a tissue-specific manner utilizing a defined subset of insulin-sensitive parameters involving alterations in enzyme specific activity and/or quantity.