Efficacy of dienogest in improving pain in women with endometriosis: a 12-month single-center experience.

PURPOSE: Dienogest has recently been marketed as a medical treatment for endometriosis. Given the recent introduction on the market of Dienogest, little data are available regarding its effectiveness in routine clinical practice. METHODS: The study is an observational, single-center, cohort study. Eligible was women with a surgical diagnosis of endometriosis dating back <24 months or a clinical/instrumental diagnosis of endometriosis and endometriosis-associated pelvic pain score of at least 40 mm on a 100-mm visual analog scale (VAS) at start of treatment and who had been taking Dienogest 2 mg once daily treatment at the time of study entry for no more than 30 days, consecutively observed between September 2013 to September 2014. In accordance with routine practice, women came back for clinical assessment and evaluation of pain after 1 (V1), 3 (V2), and 12 (V3) months. RESULTS: A total of 132 women were enrolled in the study. A total of 21 of the enrolled patients were released from the study during follow-up due to adverse effects. The mean pelvic pain VAS score at baseline was 8.9 (SD 1.3). The corresponding values were 6.7 (SD 3.2) and 5.7 (SD 3.7) for dyspareunia and dyschezia. The mean VAS scores progressively and significantly decreased to 0.9 (SD 1.6) for pelvic pain, 1.4 (SD 2.1) for dyspareunia and 0.2 (SD 0.9) for dyschezia, respectively, 12 months after start of treatment. CONCLUSION: This study confirms that in routine clinical practice, Dienogest 2 mg is an effective and well-tolerated treatment for endometriosis-related pain in women with endometriosis.

Depression after minor stroke: prevalence and predictors.

BACKGROUND AND PURPOSE: Post-stroke depression (PSD) is one of the most frequent complications of stroke, with a prevalence ranging 20-60%. As PSD seems to be related to stroke severity, we hypothesized that the prevalence of PSD would be lower in patients with minor stroke. METHODS: We investigated the prevalence and predictors of PSD over a 30-month follow-up period in a cohort of patients with minor ischaemic stroke (NIHSS≤5). RESULTS: We enrolled 105 patients (mean age 64.38±11.2years, M/F 69/36). PSD was diagnosed in 43 (41%) patients, 40 (93%) of whom had dysthymia; 22% of patients were already depressed at 1month. The most frequent depressive symptoms (DSs) were working inhibition, indecisiveness, and fatigability. Patients who developed PSD were less educated (P=0.044) and diabetic (P=0.006). After excluding patients that were already depressed at 1month, we performed a logistic regression model to detect predictors of PSD. Crying (P=0.012, OR 1.067, CI 0.269-4.553) and guilt (P=0.007, OR 0.037, CI 0.02ì03-0.401) at baseline were two DSs found to be significantly correlated with PSD. Higher educational level (P=0.022, OR 0.084, CI 0.010-0.698) and diabetes (P=0.007, OR 14.361, CI 2.040-101.108) were the risk factors significantly correlated with PSD. CONCLUSION: Post-stroke depression is frequent even in patients with minor stroke. Early detection of DSs might help to predict long-term development of PSD. No correlation was observed between lesion site or side and the development of PSD.

The alpha 6 beta 4 integrin and epithelial cell migration.

Although the involvement of alpha 6 beta 4, an integrin laminin receptor, in hemidesmosome organization has dominated the study of this integrin, recent studies are revealing novel functions for alpha 6 beta 4 in the migration of epithelial and carcinoma cells. The engagement of laminin by alpha 6 beta 4 can stabilize actin-rich protrusions and mediate traction forces necessary for cell movement. This integrin also has a significant impact on signaling molecules that stimulate migration and invasion, especially PI3-K and Rho GTPases. Activation of PI3-K by alpha 6 beta 4 enhances the formation of actin protrusions, and it may stimulate the function of other integrins, such as alpha 3 beta 1, that are also important for epithelial migration. Signaling through alpha 6 beta 4 may not always depend on the adhesive functions of this integrin, a possibility that has profound implications for migration and invasion because it implies that the ability of alpha 6 beta 4 to stimulate these processes is not limited to specific matrix environments.

Interferon gamma and lipopolysaccharide promote macrophage adherence to basement membrane glycoproteins.

The ability of thioglycollate (TG)-elicited peritoneal macrophages, a population of recently recruited monocytes, to adhere to the basement membrane glycoproteins laminin and type IV collagen is not a constitutive function of these cells. Adherence can be induced, however, by treatment with IFN-gamma and LPS. In general, IFN-gamma is more potent than LPS in promoting this adherence. Maximal adherence, however, is observed when IFN-gamma (greater than or equal to 5 U/ml) is used together with LPS (2.0 ng/ml). These requirements parallel the conditions needed to obtain tumoricidal activation of TG-elicited macrophages. Adherence to laminin, in the presence of these stimuli, is transient, being maximal at 8 h after their addition and diminishing with longer periods of incubation. In contrast, adherence to type IV collagen does not appear to be transient and IFN-gamma and LPS induce a more prolonged association of macrophages with this substratum.

Glycolipids of the mouse peritoneal macrophage. Alterations in amount and surface exposure of specific glycolipid species occur in response to inflammation and tumoricidal activation.

We have characterized the major glycolipid constituents of the mouse peritoneal macrophage, and have demonstrated that alterations in the amount and in the accessibility of specific glycolipid species to galactose oxidase/NaB3H4 labeling, an indicator of glycolipid surface exposure, occur in response to inflammation and as a consequence of activation to a tumoricidal state. The key findings are: (a) Asialo GM1, a major neutral glycolipid constituent of all macrophage populations examined, is accessible to galactose oxidase/NaB3H4 labeling on the surface of TG-elicited and BCG-activated macrophages but not on resident macrophages; (b) GM1 is the predominant ganglioside constituent of the mouse macrophage. Resident macrophages contain two distinct GM1 species, as determined by cholera toxin binding, while TG-elicited and BCG-activated macrophages contain an additional GM1 species. Differences in the relative amounts of these GM1 species, as well as in their accessibility to galactose oxidase/NaB3H4 labeling, exist among the macrophage populations. These observations suggest that both a chemical and spatial reorganization of surface glycolipids occurs in response to inflammation and tumoricidal activation.

Laminin receptors: achieving specificity through cooperation.

The laminins are a large family of extracellular matrix proteins that can profoundly influence development, differentiation and disease progression. The biological effects of the laminins are mediated by surface receptors that link laminin matrices to intracellular signalling pathways. Several classes of receptors, including integrins and other molecules, may cooperate to provide the specificity apparent in the diverse array of laminin-mediated phenomena. This review assesses our current understanding of laminin receptors and discusses how such receptors could recognize structural differences among the laminins and relay these differences to the cell.

The integrin alpha6beta4 functions in carcinoma cell migration on laminin-1 by mediating the formation and stabilization of actin-containing motility structures.

Functional studies on the alpha6beta4 integrin have focused primarily on its role in the organization of hemidesmosomes, stable adhesive structures that associate with the intermediate filament cytoskeleton. In this study, we examined the function of the alpha6beta4 integrin in clone A cells, a colon carcinoma cell line that expresses alpha6beta4 but no alpha6beta1 integrin and exhibits dynamic adhesion and motility on laminin-1. Time-lapse videomicroscopy of clone A cells on laminin-1 revealed that their migration is characterized by filopodial extension and stabilization followed by lamellae that extend in the direction of stabilized filopodia. A function-blocking mAb specific for the alpha6beta4 integrin inhibited clone A migration on laminin-1. This mAb also inhibited filopodial formation and stabilization and lamella formation. Indirect immunofluorescence microscopy revealed that the alpha6beta4 integrin is localized as discrete clusters in filopodia, lamellae, and retraction fibers. Although beta1 integrins were also localized in the same structures, a spatial separation of these two integrin populations was evident. In filopodia and lamellae, a striking colocalization of the alpha6beta4 integrin and F-actin was seen. An association between alpha6beta4 and F-actin is supported by the fact that alpha6beta4 integrin and actin were released from clone A cells by treatment with the F-actin- severing protein gelsolin and that alpha6beta4 immunostaining at the marginal edges of clone A cells on laminin-1 was resistant to solubilization with Triton X-100. Cytokeratins were not observed in filopodia and lamellipodia. Moreover, alpha6beta4 was extracted from these marginal edges with a Tween-40/deoxycholate buffer that solubilizes the actin cytoskeleton but not cytokeratins. Three other carcinoma cell lines (MIP-101, CCL-228, and MDA-MB-231) exhibited alpha6beta4 colocalized with actin in filopodia and lamellae. Formation of lamellae in these cells was inhibited with an alpha6-specific antibody. Together, these results indicate that the alpha6beta4 integrin functions in carcinoma migration on laminin-1 through its ability to promote the formation and stabilization of actin-containing motility structures.

Regulation of alpha 6 beta 1 integrin laminin receptor function by the cytoplasmic domain of the alpha 6 subunit.

The alpha 6 beta 1 integrin is expressed on the macrophage surface in an inactive state and requires cellular activation with PMA or cytokines to function as a laminin receptor (Shaw, L. M., J. M. Messier, and A. M. Mercurio. 1990. J. Cell Biol. 110:2167-2174). In the present study, the role of the alpha 6 subunit cytoplasmic domain in alpha 6 beta 1 integrin activation was examined. The use of P388D1 cells, an alpha 6-integrin deficient macrophage cell line, facilitated this analysis because expression of either the alpha 6A or alpha 6B subunit cDNAs restores their activation responsive laminin adhesion (Shaw, L. S., M. Lotz, and A. M. Mercurio. 1993. J. Biol. Chem. 268:11401-11408). A truncated alpha 6 cDNA, alpha 6-delta CYT, was constructed in which the human cytoplasmic domain sequence was deleted after the GFFKR pentapeptide. Expression of this cDNA in P388D1 cells resulted in the surface expression of a chimeric alpha 6-delta CYT beta 1 integrin that was unable to mediate laminin adhesion or increase this adhesion in response to PMA under normal conditions, i.e., in medium that contained physiological concentrations of Ca++ and Mg++. The alpha 6A-delta CYT transfectants adhered to laminin, however, when Ca++/Mg++ was replaced with 150 microM Mn++. We also assessed the role of serine phosphorylation in the regulation of alpha 6A beta 1 integrin function by site-directed mutagenesis of the two serine residues present in the alpha 6A cytoplasmic domain because this domain is phosphorylated on serine residues in response to stimuli that activate the laminin receptor function of alpha 6 A beta 1. Point mutations were introduced in the alpha 6A cDNA that changed either serine residue #1064 (M1) or serine residue #1071 (M2) to alanine residues. In addition, a double mutant (M3) was constructed in which both serine residues were changed to alanine residues. P388D1 transfectants which expressed these serine mutations adhered to laminin in response to PMA to the same extent as cells transfected with wild-type alpha 6A cDNA. These findings provide evidence for a novel mode of integrin regulation that is distinct from that reported for other regulated integrins (O'Toole, T. E., D. Mandelman, J. Forsyth, S. J. Shattil, E. F. Plow, and M. H. Ginsberg. 1991. Science (Wash. DC). 254:845-847. Hibbs, M. L., H. Xu, S. A. Stacker, and T. A. Springer. 1991. Science (Wash. DC). 251:1611-1613), and they demonstrate that serine phosphorylation of the alpha 6A cytoplasmic domain is not involved in this regulation.

Macrophage interactions with laminin: PMA selectively induces the adherence and spreading of mouse macrophages on a laminin substratum.

The ability of thioglycollate (TG)-elicited mouse peritoneal macrophages to adhere to a laminin substratum has been studied. These cells do not adhere to laminin-coated (20 micrograms/ml) surfaces, but the addition of phorbol myristate acetate (PMA; 50 ng/ml) results in their rapid adherence and spreading on this substratum. TG-elicited and PMA-activated macrophages, however, can bind soluble laminin. Macrophages adhere to fibronectin-coated surfaces and tissue culture plastic without PMA stimulation, and PMA does not increase the number of cells that adhere to these surfaces. The predominant surface proteins that bind specifically to laminin-Sepharose exhibit an Mr of 67 and 36 kD, but the expression of these proteins does not increase after PMA stimulation. Laminin receptor antibodies immunoprecipitate the 67-kD protein from radiolabled surface lysates and are capable of blocking macrophage adherence to a laminin substratum. Indirect immunofluorescence microscopy indicates that PMA stimulation does not increase receptor expression, but that it may induce the aggregation of the receptor on the cell surface. PMA stimulation also promotes macrophage spreading and induces a reorganization of the actin cytoskeleton. Taken together, these data indicate the mechanism by which PMA promotes macrophage adherence to laminin does not involve increased 67-kD receptor surface expression, but that it is related to the changes in cytoskeletal and receptor surface organization that occur in response to PMA stimulation.

Protein kinase C-dependent mobilization of the alpha6beta4 integrin from hemidesmosomes and its association with actin-rich cell protrusions drive the chemotactic migration of carcinoma cells.

We explored the hypothesis that the chemotactic migration of carcinoma cells that assemble hemidesmosomes involves the activation of a signaling pathway that releases the alpha6beta4 integrin from these stable adhesion complexes and promotes its association with F-actin in cell protrusions enabling it to function in migration. Squamous carcinoma-derived A431 cells were used because they express alpha6beta4 and migrate in response to EGF stimulation. Using function-blocking antibodies, we show that the alpha6beta4 integrin participates in EGF-stimulated chemotaxis and is required for lamellae formation on laminin-1. At concentrations of EGF that stimulate A431 chemotaxis ( approximately 1 ng/ml), the alpha6beta4 integrin is mobilized from hemidesmosomes as evidenced by indirect immunofluorescence microscopy using mAbs specific for this integrin and hemidesmosomal components and its loss from a cytokeratin fraction obtained by detergent extraction. EGF stimulation also increased the formation of lamellipodia and membrane ruffles that contained alpha6beta4 in association with F-actin. Importantly, we demonstrate that this mobilization of alpha6beta4 from hemidesmosomes and its redistribution to cell protrusions occurs by a mechanism that involves activation of protein kinase C-alpha and that it is associated with the phosphorylation of the beta4 integrin subunit on serine residues. Thus, the chemotactic migration of A431 cells on laminin-1 requires not only the formation of F-actin-rich cell protrusions that mediate alpha6beta4-dependent cell movement but also the disruption of alpha6beta4-containing hemidesmosomes by protein kinase C.

RhoA function in lamellae formation and migration is regulated by the alpha6beta4 integrin and cAMP metabolism.

Clone A colon carcinoma cells develop fan-shaped lamellae and exhibit random migration when plated on laminin, processes that depend on the ligation of the alpha6beta4 integrin. Here, we report that expression of a dominant negative RhoA (N19RhoA) in clone A cells inhibited alpha6beta4-dependent membrane ruffling, lamellae formation, and migration. In contrast, expression of a dominant negative Rac (N17Rac1) had no effect on these processes. Using the Rhotekin binding assay to assess RhoA activation, we observed that engagement of alpha6beta4 by either antibody-mediated clustering or laminin attachment resulted in a two- to threefold increase in RhoA activation, compared with cells maintained in suspension or plated on collagen. Antibody-mediated clustering of beta1 integrins, however, actually suppressed Rho A activation. The alpha6beta4-mediated interaction of clone A cells with laminin promoted the translocation of RhoA from the cytosol to membrane ruffles at the edges of lamellae and promoted its colocalization with beta1 integrins, as assessed by immunofluorescence microscopy. In addition, RhoA translocation was blocked by inhibiting phosphodiesterase activity and enhanced by inhibiting the activity of cAMP-dependent protein kinase. Together, these results establish a specific integrin-mediated pathway of RhoA activation that is regulated by cAMP and that functions in lamellae formation and migration.

Release of cAMP gating by the alpha6beta4 integrin stimulates lamellae formation and the chemotactic migration of invasive carcinoma cells.

The alpha6beta4 integrin promotes carcinoma in-vasion by its activation of a phosphoinositide 3-OH (PI3-K) signaling pathway (Shaw, L.M., I. Rabinovitz, H.H.-F. Wang, A. Toker, and A.M. Mercurio. Cell. 91: 949-960). We demonstrate here using MDA-MB-435 breast carcinoma cells that alpha6beta4 stimulates chemotactic migration, a key component of invasion, but that it has no influence on haptotaxis. Stimulation of chemotaxis by alpha6beta4 expression was observed in response to either lysophosphatidic acid (LPA) or fibroblast conditioned medium. Moreover, the LPA-dependent formation of lamellae in these cells is dependent upon alpha6beta4 expression. Both lamellae formation and chemotactic migration are inhibited or "gated" by cAMP and our results reveal that a critical function of alpha6beta4 is to suppress the intracellular cAMP concentration by increasing the activity of a rolipram-sensitive, cAMP-specific phosphodiesterase (PDE). This PDE activity is essential for lamellae formation, chemotactic migration and invasion based on data obtained with PDE inhibitors. Although PI3-K and cAMP-specific PDE activities are both required to promote lamellae formation and chemotactic migration, our data indicate that they are components of distinct signaling pathways. The essence of our findings is that alpha6beta4 stimulates the chemotactic migration of carcinoma cells through its ability to influence key signaling events that underlie this critical component of carcinoma invasion.

The activation dependent adhesion of macrophages to laminin involves cytoskeletal anchoring and phosphorylation of the alpha 6 beta 1 integrin.

Macrophages require activation with either PMA (Mercurio, A. M., and L. M. Shaw. 1988. J. Cell Biol. 107:1873-1880) or interferon-gamma (Shaw, L. M., and A. M. Mercurio. 1989. J. Exp. Med. 169:303-308) to adhere to a laminin substratum. In the present study, we identified an integrin laminin receptor on macrophages and characterized cellular changes that occur in response to PMA activation that facilitate laminin adhesion. A monoclonal antibody (GoH3) that recognizes the integrin alpha 6 subunit (Sonnenberg, A., H. Janssen, F. Hogervorst, J. Calafat, and J. Hilgers. 1987. J. Biol. Chem. 262:10376-10383) specifically inhibited adhesion to laminin-coated surfaces. This antibody precipitated an alpha 6 beta 1 heterodimer (Mr 130/110 kD) from 125I surface-labeled macrophages. The amount of radiolabeled receptor on the cell surface did not increase after PMA activation. Thus, the induction of laminin adhesion cannot be attributed to de novo or increased surface expression of alpha 6 beta 1. By initially removing the Triton X-100-soluble fraction of macrophages and then disrupting the remaining cytoskeletal framework, we observed that 75% of the alpha 6 beta 1 heterodimer on the cell surface is anchored to the cytoskeleton in macrophages that had adhered to a laminin substratum in response to PMA. Significant cytoskeletal anchoring of this receptor was not observed in macrophages that had adhered to fibronectin or tissue culture plastic, nor was it seen in nonadherent cells. PMA also induced phosphorylation of the cytoplasmic domain of the alpha 6 subunit, but not the beta 1 subunit. Phosphorylated alpha 6 was localized to the cytoskeletal fraction only in macrophages plated on a laminin substratum. In summary, our results support a mechanism for the regulation of macrophage adhesion to laminin that involves specific and dynamic matrix integrin-cytoskeletal interactions that may be facilitated by integrin phosphorylation.

The integrin alpha 6 beta 4 is a laminin receptor.

In this study, the putative laminin receptor function of the alpha 6 beta 4 integrin was assessed. For this purpose, we used a human cell line, referred to as clone A, that was derived from a highly invasive, colon adenocarcinoma. This cell line, which expresses the alpha 6 beta 4 integrin, adheres to the E8 and not to the P1 fragment of laminin. The adhesion of clone A cells to laminin is extremely rapid with half-maximal adhesion observed at 5 min after plating. Adhesion to laminin is blocked by GoH3, and alpha 6 specific antibody (60% inhibition), as well as by A9, a beta 4 specific antibody (30% inhibition). Most importantly, we demonstrate that alpha 6 beta 4 binds specifically to laminin-Sepharose columns in the presence of either Mg2+ or Mn2+ and it is eluted from these columns with EDTA but not with NaCl. The alpha 6 beta 4 integrin does not bind to collagen-Sepharose, but the alpha 2 beta 1 integrin does bind. Clone A cells do not express alpha 6 beta 1 as evidenced by the following observations: (a) no beta 1 integrin is detected in beta 1 immunoblots of GoH3 immunoprecipitates; and (b) no alpha 6 beta 1 integrin is seen in GoH3 immunoprecipitates of clone A extracts that had been immunodepleted of all beta 4 containing integrin using the A9 antibody. These data establish that laminin is a ligand for the alpha 6 beta 4 integrin and that this integrin can function as a laminin receptor independently of alpha 6 beta 1.

Evidence that distinct states of the integrin alpha6beta1 interact with laminin and an ADAM.

Integrins can exist in different functional states with low or high binding capacity for particular ligands. We previously provided evidence that the integrin alpha6beta1, on mouse eggs and on alpha6-transfected cells, interacted with the disintegrin domain of the sperm surface protein ADAM 2 (fertilin beta). In the present study we tested the hypothesis that different states of alpha6beta1 interact with fertilin and laminin, an extracellular matrix ligand for alpha6beta1. Using alpha6-transfected cells we found that treatments (e.g., with phorbol myristate acetate or MnCl2) that increased adhesion to laminin inhibited sperm binding. Conversely, treatments that inhibited laminin adhesion increased sperm binding. Next, we compared the ability of fluorescent beads coated with either fertilin beta or with the laminin E8 fragment to bind to eggs. In Ca2+-containing media, fertilin beta beads bound to eggs via an interaction mediated by the disintegrin loop of fertilin beta and by the alpha6 integrin subunit. In Ca2+-containing media, laminin E8 beads did not bind to eggs. Treatment of eggs with phorbol myristate acetate or with the actin disrupting agent, latrunculin A, inhibited fertilin bead binding, but did not induce laminin E8 bead binding. Treatment of eggs with Mn2+ dramatically increased laminin E8 bead binding, and inhibited fertilin bead binding. Our results provide the first evidence that different states of an integrin (alpha6beta1) can interact with an extracellular matrix ligand (laminin) or a membrane-anchored cell surface ligand (ADAM 2).

p53 inhibits alpha 6 beta 4 integrin survival signaling by promoting the caspase 3-dependent cleavage of AKT/PKB.

Although the interaction of matrix proteins with integrins is known to initiate signaling pathways that are essential for cell survival, a role for tumor suppressors in the regulation of these pathways has not been established. We demonstrate here that p53 can inhibit the survival function of integrins by inducing the caspase-dependent cleavage and inactivation of the serine/threonine kinase AKT/PKB. Specifically, we show that the alpha6beta4 integrin promotes the survival of p53-deficient carcinoma cells by activating AKT/PKB. In contrast, this integrin does not activate AKT/PKB in carcinoma cells that express wild-type p53 and it actually stimulates their apoptosis, in agreement with our previous findings (Bachelder, R.E., A. Marchetti, R. Falcioni, S. Soddu, and A.M. Mercurio. 1999. J. Biol. Chem. 274:20733-20737). Interestingly, we observed reduced levels of AKT/PKB protein after antibody clustering of alpha6beta4 in carcinoma cells that express wild-type p53. In contrast, alpha6beta4 clustering did not reduce the level of AKT/PKB in carcinoma cells that lack functional p53. The involvement of caspase 3 in AKT/PKB regulation was indicated by the ability of Z-DEVD-FMK, a caspase 3 inhibitor, to block the alpha6beta4-associated reduction in AKT/PKB levels in vivo, and by the ability of recombinant caspase 3 to promote the cleavage of AKT/PKB in vitro. In addition, the ability of alpha6beta4 to activate AKT/PKB could be restored in p53 wild-type carcinoma cells by inhibiting caspase 3 activity. These studies demonstrate that the p53 tumor suppressor can inhibit integrin-associated survival signaling pathways.

Combined pharmacological and short-term psychodynamic psychotherapy for probable medication overuse headache: a pilot study.

We studied the effects of short-term psychodynamic psychotherapy (STPP) and pharmacological therapy in 26 consecutive patients with probable medication overuse headache (pMOH). Patients underwent a standard in-patient detoxification protocol, lasting a mean of 7 days. Eleven patients overused non-steroidal anti-inflammatory drugs (NSAIDs), five a combination of NSAIDs and triptans, four triptans, four a combination of NSAIDs, and three triptans and ergot derivates. Preventive therapy was initiated during detoxification. The STPP protocol comprised the Brief Psychodynamic Investigation (BPI) and psychoanalysis-inspired psychotherapy. All patients (groups A and B) underwent the BPI and pharmacological therapy. Half of the patients (group B) also not randomly underwent psychoanalysis-inspired psychotherapy. We found a significant interaction between time and group for headache frequency and medication intake. At 12-month follow-up, a statistically greater decrease in headache frequency and medication intake was observed in group B than in group A (P = 0.0108 and P = 0.0097, respectively). The relapse rate was much lower in group B patients at both 6 and 12 months [15.3%, odds ratio (OR) 0.11, P = 0.016, and 23%, OR 0.18, P = 0.047, respectively] than in group A. The risk of developing chronic migraine (CM) during follow-up was higher in group A than in group B at 6 (OR 2.0, P = 0.047) and 12 months (OR 2.75, P = 0.005). Our study suggests that STPP in conjunction with drug withdrawal and prophylactic pharmacotherapy relieves headache symptoms in pMOH, reducing both long-term relapses and the burden of CM.

Reciprocal regulation of RhoA and RhoC characterizes the EMT and identifies RhoC as a prognostic marker of colon carcinoma.

Understanding how RhoC expression and activation are regulated is essential for deciphering its contribution to tumorigenesis. Here, we report that RhoC expression and activation are induced by the epithelial to mesenchymal transition (EMT) of colon carcinoma. Using LIM 1863 colon cancer cells, RhoC protein expression and subsequent activation were detected coincident with the loss of E-cadherin and acquisition of mesenchymal characteristics. Several Ets-1 binding sites were identified in the RhoC promoter, and evidence was obtained using chromatin immunoprecipitation that Ets-1 can regulate RhoC expression during the EMT. Interestingly, a marked decrease in RhoA activation associated with the EMT was observed that corresponds to the increase in RhoC expression. Use of shRNA established that RhoA inhibits and RhoC promotes post-EMT cell migration, demonstrating functional significance for their coordinate regulation. To assess the importance of RhoC expression in colon cancer, immunohistochemistry was performed on 566 colorectal tumors with known clinical outcome. The level of RhoC ranged from no expression to high expression, and statistical analysis revealed that elevated RhoC expression correlates with poor outcome as well as aberrant expression and localization of E-cadherin. These data provide one mechanism for how RhoC expression is regulated in colon carcinoma and substantiate its utility as a prognostic marker.

Regulation of cellular interactions with laminin by integrin cytoplasmic domains: the A and B structural variants of the alpha 6 beta 1 integrin differentially modulate the adhesive strength, morphology, and migration of macrophages.

Several integrin alpha subunits have structural variants that are identical in their extracellular and transmembrane domains but that differ in their cytoplasmic domains. The functional significance of these variants, however, is unknown. In the present study, we examined the possibility that the A and B variants of the alpha 6 beta 1 integrin laminin receptor differ in function. For this purpose, we expressed the alpha 6A and alpha 6B cDNAs, as well as a truncated alpha 6 cDNA (alpha 6-delta CYT) in which the cytoplasmic domain sequence was deleted after the GFFKR pentapeptide, in P388D1 cells, an alpha 6 deficient macrophage cell line. Populations of stable alpha 6A, alpha 6B, and alpha 6-delta CYT transfectants that expressed equivalent levels of cell surface alpha 6 were obtained by fluorescence-activated cell sorter and shown to form heterodimers with endogenous beta 1 subunits. Upon attachment to laminin, the alpha 6A transfectants extended numerous pseudopodia. In contrast, the alpha 6B transfectants remained rounded and extended few processes. The transfectants were also examined for their ability to migrate toward a laminin substratum using Transwell chambers. The alpha 6A transfectants were three- to fourfold more migratory than the alpha 6B transfectants. The alpha 6-delta CYT transfectants did not attach to laminin in normal culture medium, but they did attach in the presence of Mn2+. The alpha 6-delta CYT transfectants migrated to a lesser extent than either the alpha 6A or alpha 6B transfectants in the presence of Mn2+. The alpha 6 transfectants differed significantly in the concentration of substratum bound laminin required for half-maximal adhesion in the presence of Mn2+:alpha 6A (2.1 micrograms/ml), alpha 6B (6.3 micrograms/ml), and alpha 6-delta CYT (8.8 micrograms/ml). Divalent cation titration studies revealed that these transfectants also differed significantly in both the [Ca2+] and [Mn2+] required to obtain half-maximal adhesion to laminin. These data demonstrate that the A and B variants of the alpha 6 cytoplasmic domain can differentially modulate the function of the alpha 6 beta 1 extracellular domain.

Traction forces mediated by alpha6beta4 integrin: implications for basement membrane organization and tumor invasion.

The integrin alpha6beta4, a laminin receptor that stabilizes epithelial cell adhesion to the basement membrane (BM) through its association with cytokeratins, can stimulate the formation and stabilization of actin-rich protrusions in carcinoma cells. An important, unresolved issue, however, is whether this integrin can transmit forces to the substrate generated by the acto-myosin system. Using a traction-force detection assay, we detected forces exerted through alpha6beta4 on either laminin-1 or on an anti-alpha6 antibody, demonstrating that this integrin can transmit forces without the need to engage other integrins. These alpha6beta4-dependent traction forces were organized into a compression machine localized to the base of lamellae. We hypothesized that the compression forces generated by alpha6beta4 result in the remodeling of BMs because this integrin plays a major role in the interaction of epithelial and carcinoma cells with such structures. Indeed, we observed that carcinoma cells are able to remodel a reconstituted BM through alpha6beta4-mediated compression forces by a process that involves the packing of BM material under the cells and the mechanical removal of BM from adjacent areas. The distinct signaling functions of alpha6beta4, which activate phosphoinositide 3-OH kinase and RhoA, also contribute to remodeling. Importantly, we demonstrate remodeling of a native BM by epithelial cells and the involvement of alpha6beta4 in this remodeling. Our findings have important implications for the mechanism of both BM organization and tumor invasion.