Idiopathic gastroparesis is associated with specific transcriptional changes in the gastric muscularis externa.

BACKGROUND: The molecular changes that occur in the stomach that are associated with idiopathic gastroparesis are poorly described. The aim of this study was to use quantitative analysis of mRNA expression to identify changes in mRNAs encoding proteins required for the normal motility functions of the stomach. METHODS: Full-thickness stomach biopsy samples were collected from non-diabetic control subjects who exhibited no symptoms of gastroparesis and from patients with idiopathic gastroparesis. mRNA was isolated from the muscularis externa and mRNA expression levels were determined by quantitative reverse transcriptase (RT)-PCR. KEY RESULTS: Smooth muscle tissue from idiopathic gastroparesis patients had decreased expression of mRNAs encoding several contractile proteins, such as MYH11 and MYLK1. Conversely, there was no significant change in mRNAs characteristic of interstitial cells of Cajal (ICCs) such as KIT or ANO1. There was also a significant decrease in mRNA-encoding platelet-derived growth factor receptor α (PDGFRα) and its ligand PDGFB and in Heme oxygenase 1 in idiopathic gastroparesis subjects. In contrast, there was a small increase in mRNA characteristic of neurons. Although there was not an overall change in KIT expression in gastroparesis patients, KIT expression showed a significant correlation with gastric emptying whereas changes in MYLK1, ANO1 and PDGFRα showed weak correlations to the fullness/satiety subscore of patient assessment of upper gastrointestinal disorder-symptom severity index scores. CONCLUSIONS AND INFERENCES: Our findings suggest that idiopathic gastroparesis is associated with altered smooth muscle cell contractile protein expression and loss of PDGFRα+ cells without a significant change in ICCs.

Smooth muscle of telokin-deficient mice exhibits increased sensitivity to Ca2+ and decreased cGMP-induced relaxation.

Cyclic nucleotides can relax smooth muscle without a change in [Ca2+]i, a phenomenon termed Ca2+ desensitization, contributing to vasodilation, gastrointestinal motility, and airway resistance. The physiological importance of telokin, a 17-kDa smooth muscle-specific protein and target for cyclic nucleotide-induced Ca2+ desensitization, was determined in telokin null mice bred to a congenic background. Telokin null ileal smooth muscle homogenates compared to wild type exhibited an approximately 30% decrease in myosin light-chain phosphatase (MLCP) activity, which was reflected in a significant leftward shift (up to 2-fold at pCa 6.3) of the Ca2+ force relationship accompanied by an increase in myosin light-chain phosphorylation. No difference in the Ca2+ force relationship occurred in telokin WT and knockout (KO) aortas, presumably reflecting the normally approximately 5-fold lower telokin content in aorta vs. ileum smooth muscle. Ca2+ desensitization of contractile force by 8-Br-cGMP was attenuated by 50% in telokin KO intestinal smooth muscle. The rate of force relaxation reflecting MLCP activity, in the presence of 50 microM 8-Br-cGMP, was also significantly slowed in telokin KO vs. WT ileum and was rescued by recombinant telokin. Normal thick filaments in telokin KO smooth muscles indicate that telokin is not required for filament formation or stability. Results indicate that a primary role of telokin is to modulate force through increasing MLCP activity and that this effect is further potentiated through phosphorylation by cGMP in telokin-rich smooth tissues.

Shear level influences resistance artery remodeling: wall dimensions, cell density, and eNOS expression.

The magnitude of shear stimulus has been shown to determine the level of growth factor expression in cell culture. However, little is known regarding what effect shear level has on specific arterial wall remodeling events in vivo. We have hypothesized that the rate of luminal diameter change and specific remodeling events within the arterial wall layers are dependent on shear level. Selective ligations were made to alter the number of microvascular perfusion units of mesenteric arteries within the same animal to approximately 50%, 200%, and 400% of control. Arterial blood flow and wall shear rate were correlated with the degree of alteration in perfusion units. Luminal diameters were decreased in 50% arteries by day 2 and increased approximately 17% and 33% respectively, in 200% and 400% arteries at day 7. The rate of diameter change was greatest in 50% and 400% arteries. Wall areas (medial +37%; intimal +18% at day 2) and cell densities (intimal +26%; adventitial +44% at day 2) were altered only in the 400% arteries. A positive correlation existed by day 2 between endothelial staining for endothelial nitric oxide synthase and shear level. The results demonstrate that shear level influences the rate of luminal expansion, specific remodeling events within each wall layer, and the degree of endothelial gene expression. A greater understanding of how shear level influences specific remodeling events within each wall layer should aid in the development of targeted therapies to manipulate the remodeling process in health and disease.

Identification of Barx2b, a serum response factor-associated homeodomain protein.

CC(A/T)(6)GG or serum response elements represent a common regulatory motif important for regulating the expression of many smooth muscle-specific genes. They are multifunctional elements that bind serum response factor (SRF) and are important for serum induction of genes, expression of muscle-specific genes, and differentiation of vascular smooth muscle cells. In the current study, a yeast two-hybrid screen was used to identify proteins from mouse intestine that interact with SRF. A novel homeodomain-containing transcription factor, called Barx2b, was identified that specifically interacts with SRF and promotes the DNA binding activity of SRF. Northern blotting, RNase protection analysis, and Western blotting revealed that Barx2b mRNA and protein are expressed in several smooth muscle-containing tissues, as well as in skeletal muscle and brain. In vitro binding studies using bacterial fusion proteins revealed that the DNA-binding domain of SRF interacts with a region of Barx2b located amino-terminal of the homeobox domain. The results of these studies support the hypothesis that interaction of SRF with different homeodomain-containing proteins may play a critical role in determining the cell-specific functions of SRF.

Telokin expression is restricted to smooth muscle tissues during mouse development.

Telokin is a 17-kDa protein with an amino acid sequence that is identical to the COOH terminus of the 130-kDa myosin light chain kinase (MLCK). Telokin mRNA is transcribed from a second promoter, located within an intron, in the 3' region of the MLCK gene. In the current study, we show by in situ mRNA hybridization that telokin mRNA is restricted to the smooth muscle cell layers within adult smooth muscle tissues. In situ mRNA analysis of mouse embryos also revealed that telokin expression is restricted to smooth muscle tissues during embryonic development. Telokin mRNA expression was first detected in mouse gut at embryonic day 11.5; no telokin expression was detected in embryonic cardiac or skeletal muscle. Expression of telokin was also found to be regulated during postnatal development of the male and female reproductive tracts. In both uterus and vas deferens, telokin protein expression greatly increased between days 7 and 14 of postnatal development. The increase in telokin expression correlated with an increase in the expression of several other smooth muscle-restricted proteins, including smooth muscle myosin and alpha-actin.

Smooth muscle myosin light chain kinase expression in cardiac and skeletal muscle.

The purpose of this study was to characterize myosin light chain kinase (MLCK) expression in cardiac and skeletal muscle. The only classic MLCK detected in cardiac tissue, purified cardiac myocytes, and in a cardiac myocyte cell line (AT1) was identical to the 130-kDa smooth muscle MLCK (smMLCK). A complex pattern of MLCK expression was observed during differentiation of skeletal muscle in which the 220-kDa-long or "nonmuscle" form of MLCK is expressed in undifferentiated myoblasts. Subsequently, during myoblast differentiation, expression of the 220-kDa MLCK declines and expression of this form is replaced by the 130-kDa smMLCK and a skeletal muscle-specific isoform, skMLCK in adult skeletal muscle. These results demonstrate that the skMLCK is the only tissue-specific MLCK, being expressed in adult skeletal muscle but not in cardiac, smooth, or nonmuscle tissues. In contrast, the 130-kDa smMLCK is ubiquitous in all adult tissues, including skeletal and cardiac muscle, demonstrating that, although the 130-kDa smMLCK is expressed at highest levels in smooth muscle tissues, it is not a smooth muscle-specific protein.

Hepatocyte nuclear factor-3 homologue 1 (HFH-1) represses transcription of smooth muscle-specific genes.

Results show that smooth muscle-specific promoters represent novel downstream targets of the winged helix factor hepatocyte nuclear factor-3 homologue 1 (HFH-1). HFH-1 strongly represses telokin promoter activity when overexpressed in A10 vascular smooth muscle cells. HFH-1 was also found to repress transcription of several other smooth muscle-specific promoters, including the SM22alpha promoter. HFH-1 inhibits telokin promoter activity, by binding to a forkhead consensus site located within an AT-rich region of the telokin promoter. The DNA-binding domain alone was sufficient to mediate inhibition, suggesting that binding of HFH-1 blocks the binding of other positive-acting factors. HFH-1 does not disrupt serum response factor binding to an adjacent CArG box within the telokin promoter, implying that HFH-1 must compete with other unidentified trans-activators to mediate repression. The localization of HFH-1 mRNA to the epithelial cell layer of mouse bladder and stomach implicates HFH-1 in repressing telokin expression in epithelial cells. This suggests that cell-specific expression of telokin is likely mediated by both positive-acting factors in smooth muscle cells and negative-acting factors in nonmuscle cell types. We propose a model in which the smooth muscle specificity of the telokin promoter is regulated by interactions between positive- and negative-acting members of the hepatocyte nuclear factor-3/forkhead family of transcription factors.

Targeted expression of SV40 large T-antigen to visceral smooth muscle induces proliferation of contractile smooth muscle cells and results in megacolon.

Many pathological conditions result from the proliferation and de-differentiation of smooth muscle cells leading to impaired contractility of the muscle. Here we show that targeted expression of SV40 large T-antigen to visceral smooth muscle cells in vivo results in increased smooth muscle cell proliferation without de-differentiation or decreased contractility. These data suggest that the de-differentiation and proliferation of smooth muscle cells, seen in many pathological states, may be independently regulated. In the T-antigen transgenic mice the increased smooth muscle cell proliferation results in thickening of the distal colon. Consequently the distal colon becomes hyper-contractile and impedes the flow of digesta through the colon resulting in enlargement of the colon oral to the obstruction. These transgenic mice thus represent a novel model of megacolon that results from increased smooth muscle cell proliferation rather than altered neuronal innervation.

A 310-bp minimal promoter mediates smooth muscle cell-specific expression of telokin.

A cell-specific promoter located in an intron of the smooth muscle myosin light chain kinase gene directs transcription of telokin exclusively in smooth muscle cells. Transgenic mice were generated in which a 310-bp rabbit telokin promoter fragment, extending from -163 to +147, was used to drive expression of simian virus 40 large T antigen. Smooth muscle-specific expression of the T-antigen transgene paralleled that of the endogenous telokin gene in all smooth muscle tissues except uterus. The 310-bp promoter fragment resulted in very low levels of transgene expression in uterus; in contrast, a transgene driven by a 2.4-kb fragment (-2250 to +147) resulted in high levels of transgene expression in uterine smooth muscle. Telokin expression levels correlate with the estrogen status of human myometrial tissues, suggesting that deletion of an estrogen response element (ERE) may account for the low levels of transgene expression driven by the 310-bp rabbit telokin promoter in uterine smooth muscle. Experiments in A10 smooth muscle cells directly showed that reporter gene expression driven by the 2.4-kb, but not 310-bp, promoter fragment could be stimulated two- to threefold by estrogen. This stimulation was mediated through an ERE located between -1447 and -1474. Addition of the ERE to the 310-bp fragment restored estrogen responsiveness in A10 cells. These data demonstrate that in addition to a minimal 310-bp proximal promoter at least one distal cis-acting regulatory element is required for telokin expression in uterine smooth muscle. The distal element may include an ERE between -1447 and -1474.

Telokin expression in A10 smooth muscle cells requires serum response factor.

Telokin transcription is initiated from a smooth muscle-specific promoter located in an intron of the smooth muscle myosin light chain kinase gene. We have previously identified a 310-base pair fragment of the promoter that mediates A10 smooth muscle cell-specific expression of telokin. In the current study, telokin-luciferase reporter gene assays in A10 cells and REF52 nonmuscle cells revealed that the promoter region between -81 and +80 contains the regulatory elements required to mediate the in vitro cell specificity of the promoter. Several positive-acting elements, including an E box, myocyte enhancer factor 2 (MEF2)-TATA box, and CArG-serum response element, were identified within this region. Telokin transcription in A10 smooth muscle cells requires all three transcription initiation sites and an AT-rich sequence between -71 and -62 that includes a TATA box. MEF2 interacts with the AT-rich region with low affinity; however, MEF2 binding is not required for transcriptional activity in A10 cells. Binding of serum response factor (SRF) to a CArG element proximal to the TATA sequence is also critical for high levels of transcription in A10 cells. Together these data suggest that an AT-rich motif, acting in concert with SRF and an unusual transcription initiation mechanism, is required for the cell-specific expression of the telokin promoter in A10 smooth muscle cells.

Telokin expression is mediated by a smooth muscle cell-specific promoter.

The carboxy terminus of the smooth muscle myosin light chain kinase (smMLCK) is expressed as an independent protein, telokin. Western and Northern blotting analyses demonstrated that telokin protein and mRNA are expressed at high levels only in adult and embryonic smooth muscle tissues and cells. In vitro transfection assays in A10 smooth muscle cells identified a functional promoter located in an intron in the 3' region of the smMLCK gene that directs the smooth muscle cell-specific transcription of telokin. To test the cell specificity of the telokin promoter in vivo, transgenic mice were generated in which the telokin promoter was used to drive expression of SV40 large T-antigen. Expression of T-antigen in the transgenic mice paralleled that of the endogenous telokin gene. High levels of T-antigen expression were observed in smooth muscle tissues of the digestive, urinary, and reproductive tracts, with lower levels of expression in airway and vascular smooth muscle. Expression was restricted to smooth muscle cells, with no expression detected in any other cell type.

Expression of a novel myosin light chain kinase in embryonic tissues and cultured cells.

A novel, 208-kDa myosin light chain kinase (MLCK) distinct from smooth muscle and non-muscle MLCK has been identified by cross-reaction to two antibodies raised against smooth muscle MLCK. Additional antibodies directed against the amino and carboxyl termini of the smooth muscle MLCK do not react with the 208-kDa MLCK, suggesting these regions are distinct. 208-kDa MLCK phosphorylates 20-kDa myosin light chains in a Ca2+/calmodulin-dependent manner, consistent with it being a member of the MLCK family. Expression of 208-kDa MLCK and smooth muscle MLCK appears to be inversely regulated, with 208-kDa MLCK being most abundant during early development and declining at birth. In contrast, expression of smooth muscle MLCK is relatively low early during development and increases to become the predominant MLCK detected in all adult smooth and non-muscle tissues. The developmental expression pattern of the 208-kDa MLCK suggests this form be named, embryonic MLCK.

Photoaffinity labeling of a peptide substrate to myosin light chain kinase.

The substrate binding properties of skeletal muscle myosin light chain kinase were investigated with a synthetic peptide containing the photoreactive amino acid p-benzoylphenylalanine (Bpa) incorporated amino-terminal of the phosphoacceptor serine (BpaKKRAARATSNVFA). When photolyzed at 350 nm, the peptide was cross-linked stoichiometrically to myosin light chain kinase in a Ca2+/calmodulin-dependent manner. Peptide incorporation into kinase inhibited light chain phosphorylation, and the loss of kinase activity was proportional to the extent of peptide incorporated. After peptide I was incorporated into myosin light chain kinase, it was partially phosphorylated in the absence of Ca2+/calmodulin. The extent of phosphorylation increased in the presence of Ca2+/calmodulin. The cross-linked photoadduct was digested, labeled peptides were purified by high performance liquid chromatography, and sites of covalent modification were determined by amino acid sequencing and analysis. The covalent modification in the catalytic core occurred on Ile-373 (66%) and in a peptide containing residues Asn-422 to Met-437 (14%), respectively. Lys-572 in the autoinhibitory region accounted for 20% of the incorporated label. The coincident covalent modification of the autoinhibitory domain suggests that it is located near the catalytic site. Based upon a model of the catalytic core, the substrate peptide is predicted to bind in the cleft between the two lobes of the kinase. The orientation of the substrate peptide on myosin light chain kinase is similar to the orientation of the substrate recognition fragment, but not the high affinity binding fragment, of inhibitor peptide of cAMP-dependent protein kinase in the catalytic subunit of the cAMP-dependent protein kinase.

Structural requirements for phosphorylation of myosin regulatory light chain from smooth muscle.

Site-directed and chimeric mutations of myosin regulatory light chains were used to identify residues important for phosphorylation of Ser19 by smooth muscle myosin light chain kinase. Arg16 and hydrophobic residues C-terminal of Ser19 in smooth muscle light chain were important substrate determinants in the intact protein. However, changes in the kinetic properties with mutations in the light chain were substantially smaller than results reported with structurally similar synthetic peptide substrates. These results together with the low Vmax value for short peptide substrates containing the consensus phosphorylation sequence suggest that there may be additional sites of interactions between the kinase and protein substrate. Chimeras of skeletal and smooth muscle light chains were constructed with exchanges at the N terminus and subdomains I, II, III, and IV. Analysis of results obtained on the kinetic properties for phosphorylation showed that subdomains I and II contribute to high Vmax values. Thus, a region distant from the consensus phosphorylation sequence in smooth muscle light chain is also an important substrate determinant for myosin light chain kinase.

A molecular mechanism for autoinhibition of myosin light chain kinases.

It is postulated that basic residues within the inhibitory region of myosin light chain kinase (MLCK) bind acidic residues within the catalytic core to maintain the kinase in an inactive form. In this study, we identified residues within the catalytic cores of the skeletal and smooth muscle MLCKs that may bind basic residues in inhibitory region. Acidic residues within the catalytic core of the rabbit skeletal and smooth muscle MLCKs were mutated and the kinetic properties of the mutant kinases determined. Mutation of 6 and 8 acidic residues in the skeletal and smooth muscle MLCKs, respectively, result in mutant MLCKs with decreases in KCaM (the concentration of calmodulin required for half-maximal activation of myosin light chain kinase) value ranging from 2- to 100-fold. Two inhibitory domain binding residues identified in each kinase also bind a basic residue in light chain substrate. The remaining mutants all have wild-type Km values for light chain. The predicted inhibitory domain binding residues are distributed in a linear fashion across the surface of the lower lobe of the proposed molecular model of the smooth muscle MLCK catalytic core. As 6 of the inhibitory domain binding residues in the smooth muscle MLCK are conserved in other Ca2+/calmodulin-dependent protein kinases, the structural basis for autoinhibition and activation may be similar.

Substrate specificity of myosin light chain kinases.

Skeletal muscle myosin light chain kinase can phosphorylate myosin light chains isolated from skeletal or smooth muscle. In contrast, smooth muscle myosin light chain kinase specifically phosphorylates light chains isolated from smooth muscle. In this study, we have identified residues within the rabbit smooth and skeletal muscle myosin light chain kinases which may interact with the basic residues that are important substrate determinants in the light chains. Mutation of aspartic acid 270 amino-terminal of the catalytic core of the skeletal muscle myosin light chain kinase increased the Km value for both smooth and skeletal muscle light chains. Although deletions of the analogous region of the smooth muscle myosin light chain kinase (residues 663-678) markedly increased the Km value for light chain, mutation of any single acidic residue within this region did not have a similar effect. Mutation of single residues within the catalytic core of the skeletal muscle (E377 and E421) and smooth muscle (E777 and E821) myosin light chain kinases increased Km values for the smooth muscle light chain at least 35- and 100-fold, respectively. It is proposed that these residues may form ionic interactions with the arginine that is 3 residues amino-terminal of the phosphorylatable serine in the smooth muscle light chain.

Identification of basic residues involved in activation and calmodulin binding of rabbit smooth muscle myosin light chain kinase.

It is postulated that basic residues in the regulatory region of myosin light chain kinase are important for conferring autoinhibition by binding to the catalytic core. To investigate this proposal, 10 basic amino acids within the regulatory region of rabbit smooth muscle myosin light chain kinase (Lys961-Lys979) were replaced either singularly or in combination with acidic or nonpolar residues by site-directed mutagenesis. All active mutant kinases were dependent on Ca2+/calmodulin for catalytic activity. None of the mutants was active in the absence of Ca2+/calmodulin, suggesting that the autoinhibitory region has not been defined completely. Charge reversal mutants at Arg974, Arg975, and Lys976 resulted in loss of high affinity binding of calmodulin and increased the concentration of calmodulin required for half-maximal activation (KCaM). The charge reversal mutant at Lys979 also increased KCaM but to a lesser extent. Charge reversal mutants at Lys965 and Arg967 resulted in an inactive myosin light chain kinase that could not be proteolytically activated. When these residues were mutated to Ala, the expressed kinase was dependent upon Ca2+/calmodulin for activity and exhibited a decrease in KCaM. Charge reversal mutants in Lys961 and Lys962 also had decreased KCaM values. These basic residues amino-terminal of the calmodulin binding domain may play an important role in the activation of the kinase.

Biochemical properties of chimeric skeletal and smooth muscle myosin light chain kinases.

The molecular and biochemical properties of myosin light chain kinases from chicken skeletal and smooth muscle were investigated by recombinant DNA techniques. Deletion of the amino-terminal region of either the smooth or skeletal muscle myosin light chain kinase resulted in a decrease in Vmax with no significant change in Km values for light chain substrates. Skeletal/smooth muscle chimeric kinases were inactive when a 65-residue region amino-terminal of the catalytic core was exchanged between the two forms. Changing alanine 494 to glutamic acid within this region in the chicken skeletal muscle myosin light chain kinase increased the Km values for light chains 10-fold. These results are consistent with the hypothesis that the region amino-terminal of the catalytic core in myosin light chain kinases is involved in light chain recognition. A skeletal muscle kinase which contained the smooth muscle calmodulin binding domain remained regulated by Ca2+/calmodulin. Thus, the calmodulin binding domains of smooth and skeletal muscle myosin light chain kinases share structural elements necessary for regulation.