The expression and significance of lncRNA HOST2 and microRNA let-7b in HPV-positive cervical cancer tissues and cell lines.

OBJECTIVE: This study attempted to investigate the expression and significance of lncRNA HOST2 (human ovarian cancer-specific transcript 2) and microRNA let-7b in human papillomavirus (HPV)-positive cervical cancer (CC) tissues and cell lines. PATIENTS AND METHODS: The expression of levels of HOST2 and let-7b were detected by qRT-PCR in HPV-positive CC tissues and cell lines. The HPV-positive CaSki and HeLa cells were divided into the Blank, NC, pcDNA3.0-HOST2, siHOST2, let-7b mimic, and pcDNA3.0-HOST2+let-7b mimic groups. Dual-luciferase reporter gene assay was employed to verify the targeting relationship between HOST2 and let-7b, MTT and flow cytometry to determinate cell proliferation and apoptosis, and wound-healing and transwell assays to evaluate cell migration and invasion capabilities. RESULTS: HOST2 was up-regulated but let-7b was down-regulated in HPV-positive CC tissues and cells. Dual-luciferase reporter gene assay confirmed the targeting relationship between HOST2 and let-7b. Over-expressed HOST2 reduced let-7b expression, promoted proliferation migration and invasion and inhibited the apoptosis of CaSki and HeLa cells; however, silencing HOST2 or overexpressing let-7b enhanced the expression of let-7b, inhibited proliferation migration and invasion, and promoted the apoptosis of CaSki and HeLa cells, and let-7b mimic could reverse the promoting effect of HOST2 on the growth of CC cells. CONCLUSIONS: HOST2 was upregulated in HPV-positive CC tissues and cells, which could promote the proliferation, migration and invasion, but inhibit the apoptosis of HPV-positive CC cells via inhibition of let-7b.

Increased expression of long noncoding RNA HMMR-AS1 in epithelial ovarian cancer: an independent prognostic factor.

OBJECTIVE: Evidence has indicated that long noncoding RNA (lncRNAs) may have significant roles in cancer. In this study, we aimed to investigate the expression pattern and prognostic value of a noncoding RNA named as HMMR antisense RNA 1 (HMMR-AS1) in epithelial ovarian cancer (EOC). PATIENTS AND METHODS: Differences in the expression of HMMR-AS1 between EOC and matched normal tissues were analyzed using RT-PCR. The correlation between HMMR-AS1 levels and the clinicopathological factors of the EOC patients was analyzed by x2-test. Kaplan-Meier analysis and Cox proportional hazards regression models were explored to reveal the correlations of HMMR-AS1 expression with survival of patients. RESULTS: HMMR-AS1 was significantly upregulated in human EOC tissues compared with adjacent normal tissues (p < 0.01). Clinicopathologic analysis revealed that high expression of HMMR-AS1 was associated with advanced FIGO stage (p = 0.013) and positive lymphatic metastasis (p = 0.010). Moreover, patients with higher HMMR-AS1 expression displayed shorter overall survival time (p = 0.0075) and progression-free survival time (p = 0.0013) than those with lower HMMR-AS1 expression. More importantly, multivariate analysis suggested that high expression of HMMR-AS1 was an independent prognostic indicator for EOC patients. CONCLUSIONS: Our data suggested that HMMR-AS1 may be considered a novel prognostic factor in EOC and a specific diagnostic indicator for patients with EOC.

Paracrine communication regulates adrenocorticotropin secretion.

Local communication among cells of the anterior pituitary appears to play an important role in the regulation of ACTH secretion. Dissociated pituitary cells were plated as a monolayer at decreasing concentrations of cells (increasing the distance between cells and, thus, decreasing their potential interactions), and ACTH secretion was measured from individual corticotropes using a specific reverse hemolytic plaque assay. There was a critical intercell distance above which significant changes in the number of CRF-responsive corticotropes were observed. Provided that this critical distance was not exceeded the number of secretory corticotropes in response to CRF (10 nM) was relatively constant, thereby defining a fraction of corticotropes that was robustly CRF responsive. In contrast, when this critical distance between cells was exceeded, the number of CRF-responsive corticotropes progressively increased to almost double their original number, thereby defining a second fraction of CRF-responsive corticotropes that was previously repressed. These observations suggest the presence of a paracrine factor that profoundly inhibits CRF-stimulated ACTH secretion from a repressed fraction of corticotropes. Further independent studies confirmed and extended these observations. We identified the cellular source of the inhibitory factor as the robustly CRF-responsive fraction of corticotropes. Pituitary cells were identified by reverse hemolytic plaque assay and then destroyed using a laser photoablation procedure that did not compromise the remaining cells. The pituitary cells were separated by a distance at which the inhibitory factor was fully effective. Destruction of the cellular source of the paracrine inhibition would, therefore, allow secretion from the previously repressed fraction of corticotropes. Accordingly, when robustly CRF-responsive corticotropes were destroyed, a significant number of previously repressed corticotropes appeared in a second assay. Destruction of somatotropes or a cell adjacent to a robustly CRF-responsive corticotrope did not alter the number of CRF-stimulated corticotropes among the remaining cells. We conclude that a paracrine factor liberated by the robustly CRF-responsive corticotropes inhibits ACTH secretion from the repressed fraction of corticotropes. The robustly CRF-responsive corticotropes appear unresponsive to the effects of the factor, and the repressed corticotropes are unlikely to secrete it. A role of this paracrine communication is to hold corticotropes in reserve and, therefore, prevent the severe depletion of hormone. This form of paracrine communication may be a specialized adaption among cells where the physiological setting demands robust secretory responses to multiple stimuli. The experimental paradigms developed here may be extremely useful for 1) screening potential paracrine factors and 2) determining whether the secretion of the paracrine factor is regulated by adrenal or hypothalamic hormones.

Distinct classes of corticotropes mediate corticotropin-releasing hormone- and arginine vasopressin-stimulated adrenocorticotropin release.

ACTH release from the anterior pituitary gland is principally driven by the two hypothalamic hormones, corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP). Using the reverse hemolytic plaque assay, we have compared the effects of CRH and AVP on ACTH release from individual, dispersed pituitary cells. A small percent (0.36 +/- 0.06%) of pituitary cells formed plaques when exposed to medium alone. AVP caused 3.44 +/- 0.10% of cells to form plaques (P less than 0.01 compared with medium alone), CRH produced 4.85 +/- 0.20% plaque-forming cells (P less than 0.01 compared with AVP), and the combination of CRH and AVP produced a still greater percent of plaque-forming cells (5.80 +/- 0.20%, P less than 0.01 compared with CRH alone). A double reverse hemolytic plaque assay was then employed to examine whether some cells formed plaques only in the presence of one or other secretagogue. Using this technique we found clear evidence of cells that formed plaques in response to CRH but not AVP (P less than 0.005); CRH or AVP (P less than 0.0001), and CRH and AVP (P less than 0.05). There was no evidence of a corticotrope forming a plaque with AVP but not CRH (P = 0.52). Thus there appears to be functionally distinct classes of corticotropes. These findings have important implications for our understanding of the relative responsiveness of the pituitary to hypothalamic secretagogues and provide a new physiological perspective on recent reports of stress-specific hypothalamic responses regulating ACTH release.

Gene structure and expression of phospholemman in mouse.

Phospholemman (PLM) is a small transmembrane cardiac protein that is the major sarcolemmal substrate for phosphorylation in response to adrenergic stimulation. PLM likely plays a role in muscle contractility and cell volume regulation through its function as a channel or a channel regulator. We are the first to describe the structure of the PLM gene and to demonstrate PLM cDNA splice variants. We cloned the murine PLM cDNA and used it as a probe to isolate the gene from a 129/SvJ genomic library. The gene contains seven introns and eight exons. The coding sequence is interrupted by five introns; the 5' untranslated region by two. Using rapid amplification of 5' cDNA ends we identified transcription start sites and four splice variants of the 5' untranslated domain. There was no TATA box or CAAT box in the putative promoter regions. The gene has several stretches of dinucleotide repeats. The 3' untranslated domains of mouse PLM cDNA clones show sequence differences not accounted for by alternative splicing. Mouse PLM shares 93, 83 and 80% amino acid identity with rat, dog, and human PLMs, respectively. Tissue expression of murine PLM parallels that in other species, being highest in heart, skeletal muscle, and liver.

Snake venom metalloproteinases: structure, function and relationship to the ADAMs family of proteins.

A large number of zinc metalloproteinases of varying mol. wts and biological functions has been isolated from crotalid and viperid venoms. Over the past few years, structural studies on these proteinases have suggested their organization into four classes, P-I to P-IV. These proteinases are synthesized in the venom gland as zymogens which are subsequently processed to the active form. The signal and pro-sequences of the proteins are highly conserved. Within the pro-domain lies a consensus sequence which probably functions in a manner similar to the cysteine switch in mammalian collagenases. The proteinase domain is represented by two forms: a two-disulfide and a three-disulfide structure. Crystallographic and modeling studies suggest that the two forms share very similar tertiary structures. The larger venom metalloproteinases (P-II, III and IV) have additional domains on the carboxy side of the proteinase domain. The additional domains that have been identified include disintegrin and disintegrin-like domains, a high-cysteine domain and a lectin-binding domain. It appears that these non-enzymatic domains function to modulate the biological properties of the proteinases. Recently, a family of homologues of the venom zinc metalloproteinases has been described from a variety of organisms including mammals, reptiles and invertebrates. This family of proteins has been termed the ADAMs, for A Disintegrin-like And Metalloproteinase-containing protein. They differ from the venom proteinases in that some of them may not have proteolytic activity. In addition to the domain structure described for the P-III class of venom proteins, the ADAMs have an epidermal growth factor-like domain, a transmembrane domain and a cytoplasmic domain. A description of venom metalloproteinase structure will be outlined in this review, along with the similarities and differences among the venom proteins and the ADAMs family of proteins.

Tregs are dysfunctional in vivo in a spontaneous murine model of Crohn's disease.

Although regulatory T cells (Tregs) have been implicated in inflammatory bowel disease, Tregs from Crohn's disease (CD) patients are increased in number and function normally in vitro. To clarify this disparity, we studied Treg function in vivo using a spontaneous model of CD-like ileitis. We first administered anti-CD25-depleting antibodies to SAMP1/YitFc (SAMP) mice to assess ileitis; mesenteric lymph node cells were then transferred into SCID (severe combined immunodeficient) recipients to induce colitis. CD25 depletion increased the severity of both spontaneous ileitis and adoptively transferred colitis. Interestingly, a second transfer of CD4(+)CD25(+) cells from untreated AKR control mice was able to ameliorate the induced colitis, whereas CD4(+)CD25(+) cells from untreated SAMP mice were not, suggesting a functional abnormality in SAMP Tregs. Anti-CD25 treatment in SAMP mice also induced proliferation of CD25(-)Foxp3(+) Tregs, which had a proinflammatory intestinal T helper type 1/ T helper type 2 (Th1/Th2) effector phenotype. These studies demonstrate Treg dysfunction in a spontaneous model of CD-like ileitis.

Corticotropin-releasing factor, but not arginine vasopressin, stimulates concentration-dependent increases in ACTH secretion from a single corticotrope. Implications for intracellular signals in stimulus-secretion coupling.

The two fundamental parameters of corticotropin (ACTH) secretion are the number of secreting corticotropes and the amount of ACTH secreted by each cell. We have measured these parameters in rat corticotropes in response to increasing concentrations of corticotropin-releasing factor (CRF) or arginine vasopressin (AVP). Increasing concentrations of AVP stimulated more corticotropes to secrete, while the amount of ACTH each cell secreted remained relatively fixed (nongraded secretory response). Conversely, increasing concentrations of CRF stimulated more ACTH secretion per cell (graded secretory response), while the number of secretory cells remained relatively constant. When viewed from the perspective of a single corticotrope, it was clear that CRF and AVP induced completely distinct specific responses. We have previously shown, and provide further evidence here, that secretory responses to CRF or AVP occur in the same cell. It is therefore apparent that a single corticotrope is able to generate either a graded, or a nongraded secretory response. We have also considered the potential intracellular changes that must direct graded or nongraded secretion. It is generally accepted that CRF stimulates activation of adenylate cyclase, whereas AVP activates phosphoinositidase in pituitary corticotropes. Our findings, and others surveyed here, suggest that the activation of adenylate cyclase results in graded secretion, while the activation of phosphoinositidase induces the nongraded secretion. Graded or nongraded secretion may therefore be linked to specific second messengers. It is hypothesized that the inositol 1,4,5-trisphosphate-mediated release of an intracellular Ca2+ store constitutes a mechanism whereby phosphoinositidase-coupled hormones set in motion the nongraded secretory response. These findings suggest novel functions for individual second messengers.

Dominance of G(s) in doubly G(s)/G(i)-coupled chimaeric A(1)/A(2A) adenosine receptors in HEK-293 cells.

A(1) adenosine receptors inhibit adenylate cyclase by activating G(i)/G(o), whereas A(2A) receptors activate G(s). We examined how regions of A(1) and A(2A) receptors regulate coupling to G-proteins by constructing chimaeras in which the third intracellular loops (3ICL or L) and/or the C-termini (or T) were switched. Pertussis toxin (PTX) was used in membrane radioligand binding assays to calculate the fraction of recombinant receptors coupled to G(i)/G(o) and in whole cells to differentially influence agonist-stimulated cAMP accumulation. Switching A(1)/A(2A) 3ICL domains results in receptors that maintain binding selectivity for ligands but are doubly coupled. Receptor chimaeras with an A(1) 3ICL sequence (A(2A)/A(1)L or A(2A)/A(1)LT) respond to agonist stimulation with elevated cAMP despite being coupled predominantly to G(i)/G(o). These chimaeras have basal cAMP levels lower than those of wild-type A(2A) receptors, similar to wild-type A(1) receptors. The A(1) C-terminus modulates the coupling of receptors with A(1) 3ICL such that A(2A)/A(1)LT is better coupled to G(i)/G(o) than A(2A)/A(1)L. The C-terminus has little impact on coupling to receptors containing A(2A) 3ICL sequence. Our results show that the C-terminus sequence selectively facilitates coupling to G(i)/G(o) mediated by A(1) 3ICL and not by other intracellular domains that favour G(i) coupling. The C-terminus sequence has little or no effect on coupling to G(s). For doubly G(s)/G(i)-coupled adenosine receptors in HEK-293 cells, G(s)-mediated stimulation predominates over G(i)/G(o)-mediated inhibition of adenylate cyclase. We discuss the signalling consequences of simultaneously activating opposing G-proteins within single cells.

Sequence and biological activity of catrocollastatin-C: a disintegrin-like/cysteine-rich two-domain protein from Crotalus atrox venom.

In this study we report on the isolation and biological characterization of a 23.6-kDa protein from the venom of the western diamondback rattlesnake, Crotalus atrox. Primary structural analysis shows the protein to be composed of a spacer/disintegrin-like domain and a cysteine-rich domain. The sequence is identical to the same carboxy-terminal domains found in the C. atrox metalloproteinase, catrocollastatin, and hence we termed the protein catrocollastatin-C. We estimate that catrocollastatin-C represents at least 0.5% of the total protein in C. atrox venom. The protein is an inhibitor of collagen-stimulated but not ADP-stimulated platelet aggregation. Reduction and alkylation of catrocollastatin-C causes a loss of platelet aggregation inhibitor activity. A synthetic, cyclic peptide designed from the catrocollastatin-C disintegrin-like domain has potent platelet aggregation inhibitory activity. This suggests that the corresponding region in the disintegrin-like domain of the protein is at least partially responsible for the inhibition of platelet aggregation previously reported for the protein. These studies underscore the biochemical and functional complexity of crotalid snake venoms due to differential proteolytic processing of precursor proteins and how the processed precursor fragments may contribute to the observed pathological effects of the venom.

cDNA sequences for four snake venom metalloproteinases: structure, classification, and their relationship to mammalian reproductive proteins.

Presented here are four new cDNA sequences for hemorrhagic metalloproteinases from Crotalus atrox venom, hemorrhagic toxins a, b, c, and d. Comparison of the translated open reading frames to the mature protein sequences gives evidence for post-translational processing at both the amino and carboxyl termini. This comparison is also the basis for a new classification system for these precursors, based on their different sizes. Protein sequences in the zymogen region support the hypothesis of a cysteine-switch type mechanism of maintaining latency. The coordination geometry around the active site zinc ion is discussed. The relationship between these venom metalloproteinases and a family of mammalian reproductive proteins is also supported by these sequences. The cysteine pattern of the carboxyl-terminal domain of the largest proteinase, hemorrhagic toxin a, is compared to other venom proteinases and to the mammalian proteins, showing both striking similarities and subtle differences. It would appear that these hemorrhagic toxins have resulted from deletions and subsequent divergence from a larger ancestor, one they may have shared with the aforementioned mammalian reproductive proteins.

Expression, activation, and processing of the recombinant snake venom metalloproteinase, pro-atrolysin E.

The expression in human embryonic kidney (HEK 293) cells of the recombinant zymogen form (pro-) of the Crotalus atrox hemorrhagic metalloproteinase, atrolysin E, is presented. The nascent protein is comprised of pre-, pro-, proteinase-, spacer-, and disintegrin domains. The biochemical characterization of the recombinant zymogen is described along with its activation by C. atrox crude venom and other hemorrhagic toxins. Unlike the zymogen forms of the matrix metalloproteinases, pro-atrolysin E is not activated by the organomercurial, (4-aminophenyl)mercuric acetate. Pro-atrolysin E could be enzymatically activated by C. atrox crude venom, PMSF-inhibited crude venom, atrolysin A, and atrolysin E itself. There is no evidence of autoactivation. Using two polyclonal antibodies directed against the proteinase domain and the disintegrin domain of atrolysin E, the proteolytic processing of the recombinant protein by atrolysin A was followed. The first cleavage of pro-atrolysin E by atrolysin A removes the pro-domain. The second proteolysis step removes the disintegrin domain to produce the proteinase/spacer protein. These studies have identified potential activators of snake venom pro-metalloproteinases in crude venom and suggest a general scheme for the activation and processing of venom pro-metalloproteinases by the endogenous, active metalloproteinases.

Isolation, sequence analysis, and biological activity of atrolysin E/D, the non-RGD disintegrin domain from Crotalus atrox venom.

Crotalid snake venom metalloproteinases often have associated with them nonproteinase domains that may be processed from the mature proteinases. Nascent atrolysin E, from the western diamondback rattlesnake, Crotalus atrox, has a metalloproteinasedomain and a non-RGD disintegrin domain that is lacking in the mature metalloproteinase. In this studywe report on the isolation, sequence analysis, andbiological activity of the 7.4-kDa atrolysin E disintegrin domain (atrolysin E/D). Atrolysin E/D represents approximately 0.2% of the total protein fromthe crude venom. The protein begins with a glycinyl residue found in the latter part of the spacer region. The sequence of atrolysin E/D is identical to thatof the non-RGD disintegrin domain of atrolysin E. The structure is termed a non-RGD disintegrin sincein lieu of the characteristic RGD sequence, a Met-Val-Asp (MVD) is found instead. Nevertheless, the protein is a potent inhibitor of both collagen- and ADP-stimulated platelet aggregation with IC50 values of 4 and 8 nM, respectively. A cyclized synthetic peptide, Ac-CRVSMVDRNDDTC-NH2, which represents the sequence of the atrolysin E/D non-RGD loop, was demonstrated to be an effective inhibitor of platelet aggregation. Therefore, this region of atrolysin E/D's structure, as in the disintegrins proper, is important for the biological activity of the protein. Thus, like the non-RGD disintegrin barbourin from Sistrurus miliarius barbouri, a RGD sequence in the context of the disintegrin protein backbone is not an absolute requirement for platelet aggregation inhibitory activity. These data underscore the biochemical and functional complexity of crotalid snake venoms due to differential proteolytic processing of the precursor metalloproteinases and exemplify how the processed fragments may contribute to the observed pathological effects of the venom.

Function of disintegrin-like/cysteine-rich domains of atrolysin A. Inhibition of platelet aggregation by recombinant protein and peptide antagonists.

Snake venom hemorrhagic metalloproteinase toxins that have metalloproteinase, disintegrin-like and cysteine-rich domains are significantly more potent than toxins with only a metalloproteinase domain. The disintegrin-like domains of these toxins differ from the disintegrin peptides found in crotalid and viperid venoms by the nature of their different disulfide bond structure and, in lieu of the disintegrins' signature Arg-Gly-Asp (RGD) integrin binding sequence, there is an XXCD disulfide-bonded cysteinyl sequence in that region. Due to these apparent differences, the contribution to the overall function of the hemorrhagic metalloproteinases by the disintegrin-like domain has been unknown. In this investigation we have expressed in insect cells the disintegrin-like/cysteine-rich (DC) domains of the Crotalus atrox hemorrhagic metalloproteinase atrolysin A and demonstrated that the recombinant protein (A/DC) can inhibit collagen- and ADP-stimulated platelet aggregation. Using synthetic peptides, we have evidence that the region of the disintegrin-like domain that is positionally analogous to the RGD loop of the disintegrins is the site responsible for inhibition of platelet aggregation. For these synthetic peptides to have significant inhibitory activity, the -RSECD- cysteinyl residue must be constrained by participation in a disulfide bond with another cysteinyl residue. The two acidic amino acids adjacent to the middle cysteinyl residue in these peptides are also important for biological activity. These studies emphasize a functional role for the disintegrin-like domain in toxins and suggest structural possibilities for the design of antagonists of platelet aggregation.

Inhibition of platelet aggregation by the recombinant cysteine-rich domain of the hemorrhagic snake venom metalloproteinase, atrolysin A.

The P-III class of venom metalloproteinases has, in addition to the proteinase domain, a disintegrin-like domain and a cysteine-rich domain. Recent evidence has shown that the nonproteinase domains of the P-III class of hemorrhagic metalloproteinases function in the inhibition of platelet aggregation by blocking essential procoagulant integrins on platelets. A specific role for the highly conserved cysteine-rich domain has yet to be described. In this study, we expressed the cysteine-rich domain from the hemorrhagic metalloproteinase atrolysin A and demonstrated its ability to inhibit collagen-stimulated platelet aggregation. Additionally, the cysteine-rich domain was shown to interact with MG-63 cells to inhibit adhesion to collagen I. These data suggest a functional role for the cysteine-rich domain of the P-III toxins in the observed coagulopathy by targeting the toxin to platelets and inhibiting collagen-stimulated platelet aggregation. These characteristics may function to synergistically increase the hemorrhagic effect of the toxins.