Differential Myosin 5a splice variants in innervation of pelvic organs.

Introduction: Myosin proteins interact with filamentous actin and translate the chemical energy generated by ATP hydrolysis into a wide variety of mechanical functions in all cell types. The classic function of conventional myosins is mediation of muscle contraction, but myosins also participate in processes as diverse as exocytosis/endocytosis, membrane remodeling, and cytokinesis. Myosin 5a (Myo5a) is an unconventional motor protein well-suited to the processive transport of diverse molecular cargo within cells and interactions with multiprotein membrane complexes that facilitate exocytosis. Myo5a includes a region consisting of six small alternative exons which can undergo differential splicing. Neurons and skin melanocytes express characteristic splice variants of Myo5a, which are specialized for transport processes unique to those cell types. But less is known about the expression of Myo5a splice variants in other tissues, their cargos and interactive partners, and their regulation. Methods: In visceral organs, neurotransmission-induced contraction or relaxation of smooth muscle is mediated by Myo5a. Axons within urogenital organs and distal colon of rodents arise from cell bodies located in the major pelvic ganglion (MPG). However, in contrast to urogenital organs, the distal colon also contains soma of the enteric nervous system. Therefore, the rodent pelvic organs provide an opportunity to compare the expression of Myo5a splice variants, not only in different tissues innervated by the pelvic nerves, but also in different subcellular compartments of those nerves. This study examines the expression and distribution of Myo5a splice variants in the MPG, compared to the bladder, corpus cavernosum of the penis (CCP) and distal colon using immunohistochemistry and mRNA analyses. Results/discussion: We report detection of characteristic Myo5a variants in these tissues, with bladder and CCP displaying a similar variant pattern but one which differed from that of distal colon. In all three organs, Myo5a variants were distinct compared to the MPG, implying segregation of one variant within nerve soma and its exclusion from axons. The expression of distinct Myo5a variant arrays is likely to be adaptive, and to underlie specific functions fulfilled by Myo5a in those particular locations.

Myosin 5a in the Urinary Bladder: Localization, Splice Variant Expression, and Functional Role in Neurotransmission.

Dysregulation of neurotransmission is a feature of several prevalent lower urinary tract conditions, but the mechanisms regulating neurotransmitter release in the bladder are not completely understood. The unconventional motor protein, Myosin 5a, transports neurotransmitter-containing synaptic vesicles along actin fibers towards the varicosity membrane, tethering them at the active zone prior to reception of a nerve impulse. Our previous studies indicated that Myosin 5a is expressed and functionally relevant in the peripheral nerves of visceral organs such as the stomach and the corpora cavernosa. However, its potential role in bladder neurotransmission has not previously been investigated. The expression of Myosin 5a was examined by quantitative PCR and restriction analyses in bladders from DBA (dilute-brown-nonagouti) mice which express a Myosin 5a splicing defect and in control mice expressing the wild-type Myosin 5a allele. Functional differences in contractile responses to intramural nerve stimulation were examined by ex vivo isometric tension analysis. Data demonstrated Myosin 5a localized in cholinergic nerve fibers in the bladder and identified several Myosin 5a splice variants in the detrusor. Full-length Myosin 5a transcripts were less abundant and the expression of splice variants was altered in DBA bladders compared to control bladders. Moreover, attenuation of neurally-mediated contractile responses in DBA bladders compared to control bladders indicates that Myosin 5a facilitates excitatory neurotransmission in the bladder. Therefore, the array of Myosin 5a splice variants expressed, and the abundance of each, may be critical parameters for efficient synaptic vesicle transport and neurotransmission in the urinary bladder.

Expression of Myosin 5a splice variants in murine stomach.

BACKGROUND: The motor protein, Myosin 5a (Myo5a) is known to play a role in inhibitory neurotransmission in gastric fundus. However, there is no information regarding the relative expression of total Myo5a, or of its alternative exon splice variants, across the stomach. This study investigated the differential distribution of Myo5a variants expressed within distinct anatomical regions of murine stomach. METHODS: The distribution of Myo5a protein and mRNA in the stomach was assessed by immunofluorescence microscopy and fluorescent in situ hybridization. Quantitative PCR, restriction enzyme analysis, and electrophoresis were used to identify Myo5a splice variants and quantify their expression levels in the fundus, body, antrum, and pylorus. KEY RESULTS: Myo5a protein colocalized with ßIII-Tubulin in the myenteric plexus, and with synaptophysin in nerve fibers. Total Myo5a mRNA expression was lower in pylorus than in antrum, body, or fundus (p < 0.001), which expressed equivalent amounts of Myo5a. However, Myo5a splice variants were differentially expressed across the stomach. While the ABCE splice variant predominated in the antrum and body regions, the ACEF/ACDEF variants were enriched in fundus and pylorus. CONCLUSIONS AND INFERENCES: Myo5a splice variants varied in their relative expression across anatomically distinguishable stomach regions and might mediate distinct physiological functions in gastric neurotransmission.

Myosin Va plays a role in nitrergic smooth muscle relaxation in gastric fundus and corpora cavernosa of penis.

The intracellular motor protein myosin Va is involved in nitrergic neurotransmission possibly by trafficking of neuronal nitric oxide synthase (nNOS) within the nerve terminals. In this study, we examined the role of myosin Va in the stomach and penis, proto-typical smooth muscle organs in which nitric oxide (NO) mediated relaxation is critical for function. We used confocal microscopy and co-immunoprecipitation of tissue from the gastric fundus (GF) and penile corpus cavernosum (CCP) to localize myosin Va with nNOS and demonstrate their molecular interaction. We utilized in vitro mechanical studies to test whether smooth muscle relaxations during nitrergic neuromuscular neurotransmission is altered in DBA (dilute, brown, non-agouti) mice which lack functional myosin Va. Myosin Va was localized in nNOS-positive nerve terminals and was co-immunoprecipitated with nNOS in both GF and CCP. In comparison to C57BL/6J wild type (WT) mice, electrical field stimulation (EFS) of precontracted smooth muscles of GF and CCP from DBA animals showed significant impairment of nitrergic relaxation. An NO donor, Sodium nitroprusside (SNP), caused comparable levels of relaxation in smooth muscles of WT and DBA mice. These normal postjunctional responses to SNP in DBA tissues suggest that impairment of smooth muscle relaxation resulted from inhibition of NO synthesis in prejunctional nerve terminals. Our results suggest that normal physiological processes of relaxation of gastric and cavernosal smooth muscles that facilitate food accommodation and penile erection, respectively, may be disrupted under conditions of myosin Va deficiency, resulting in complications like gastroparesis and erectile dysfunction.

Upregulation of the coagulation factor VII gene during glucose deprivation is mediated by activating transcription factor 4.

BACKGROUND: Constitutive production of blood coagulation proteins by hepatocytes is necessary for hemostasis. Stressful conditions trigger adaptive cellular responses and delay processing of most proteins, potentially affecting plasma levels of proteins secreted exclusively by hepatocytes. We examined the effect of glucose deprivation on expression of coagulation proteins by the human hepatoma cell line, HepG2. METHODOLOGY/PRINCIPAL FINDINGS: Expression of coagulation factor VII, which is required for initiation of blood coagulation, was elevated by glucose deprivation, while expression of other coagulation proteins decreased. Realtime PCR and ELISA demonstrated that the relative percentage expression +/- SD of steady-state F7 mRNA and secreted factor VII antigen were significantly increased (from 100+/-15% to 188+/-27% and 100+/-8.8% to 176.3+/-17.3% respectively, p<0.001) at 24 hr of treatment. The integrated stress response was induced, as indicated by upregulation of transcription factor ATF4 and of additional stress-responsive genes. Small interfering RNAs directed against ATF4 potently reduced basal F7 expression, and prevented F7 upregulation by glucose deprivation. The response of the endogenous F7 gene was replicated in reporter gene assays, which further indicated that ATF4 effects were mediated via interaction with an amino acid response element in the F7 promoter. CONCLUSIONS/SIGNIFICANCE: Our data indicated that glucose deprivation enhanced F7 expression in a mechanism reliant on prior ATF4 upregulation primarily due to increased transcription from the ATF4 gene. Of five coagulation protein genes examined, only F7 was upregulated, suggesting that its functions may be important in a systemic response to glucose deprivation stress.

Polymorphisms in the factor VII gene and ischemic stroke in young adults.

Polymorphic configurations of the coagulation factor VII gene (F7) are associated with plasma levels of FVII antigen (FVII:Ag) and FVII coagulant activity (FVII:C). Our aim was to determine whether F7 polymorphisms influence risk of ischemic stroke in young adults. One hundred and fifty survivors of ischemic stroke before the age of 45 and an equal number of age and sex-matched controls were genotyped for five F7 polymorphisms: the -A670C transversion, -323 decanucleotide insertion (P + 10), the number (which varies between five and eight) of a 37 base pair repeat polymorphisms in intron 7 (IVS7), amino acid substitution R353Q, and +154AA insertion. 353Q, P + 10 and +154AA were demonstrated to associate with significantly decreased plasma FVII:Ag, whereas -670C and IVS7 seven or higher were associated with a tendency towards increased plasma FVII:Ag. The former three polymorphisms were significantly more common in control individuals than in patients, whereas the latter two were significantly more common in patients than in control individuals. The multiple logistic regression analysis revealed that two F7 polymorphisms, -670C and IVS7 seven or higher, are independent risk factors for ischemic stroke in young adult patients.

CCAAT/enhancer-binding protein-beta participates in insulin-responsive expression of the factor VII gene.

Expression of the human coagulation factor VII (FVII) gene by hepatoma cells was modulated in concert with levels of glucose and insulin in the culture medium. In low glucose medium without insulin, amounts of both FVII mRNA and secreted FVII protein were coordinately increased; in the presence of glucose with insulin, both were decreased. Analysis of the FVII promoter showed that these effects could be reproduced in a reporter-gene system, and a small promoter element immediately upstream of the translation start site of the gene, which mediated these effects, was identified. Mutation of this element largely abrogated the glucose/insulin-responsive change in expression of the reporter gene. Several members of the CCAAT/enhancer-binding protein family were found to be capable of binding the identified sequence element but not the mutated element. The expression of a FVII minigene directed by a segment of the native FVII promoter responded to co-expressed activating and inhibiting forms of CCAAT/enhancer-binding protein beta.

Two naturally occurring mutations on FVII gene (S363I-W364C) altering intrinsic catalytic activity.

Factor VII (FVII) requires the cleavage of an internal peptide bond and the association with tissue factor (TF) to attain its fully active FVIIa conformation. This event alone leaves FVIIa in a zymogen-like state of relatively low specific activity. The TF-induced allosteric enhancement of FVIIa's activity contributes to the procoagulant activity of the complex. We have characterized two naturally occurring mutations (S363I - W364C) on FVII gene. Both homozygous patients for each mutation have a normal FVII: Ag level associated to an undetectable FVII coagulant activity. The patient carrying the allele 364C had a more severe hemorrhagic diathesis than the S363I mutant. To understand the mechanism of these deficiency, in vitro expression analysis with further biochemical characterization of recombinant proteins of both mutants FVII-363I, FVII-364C and wild type (WTFVII) FVII constructs were done. The results recapitulated the patients' plasma data with normal Ag level and no detectable coagulant activity. The D-F-Pip-R-pNA and CH(3)SO(2)-D-CHA-A-But-R chromogenic substrates were used to evaluate the amidolytic activity of WT and mutant FVII in presence and absence of recombinant tissue factor (rTF). Binding of FVII to rTF by a solid phase binding assay was done using recombinant human rTF. The results of amidolytic assays showed that rTF enhances 28 fold the value of the specificity of constant (kcat/K(m)) in WT but no activity was detectable in either mutant constructs under any condition. The equilibrium dissociation constant of rTF-FVIIa interaction showed Kd equal to 4.4 +/- 0.2nM, 4.9 +/- 0.5nM and 6 +/- 0.9 of WT, 363I and 364C FVII forms, respectively. The K(d) values of the non activated forms were equal to 24.7 +/- 3.3, 24.4 +/- 3.1 and 20.6 +/- 4nM, respectively. These data demonstrate that, compared to the WT form, FVII-363I and FVII-364C have no significant affinity change for TF and that the detrimental effect of these two mutations is attributable to the loss of an efficient catalytic machinery in the FVII molecule causing a severe deficiency of coagulant activities.