Clinical response to linaclotide at week 4 predicts sustained response in irritable bowel syndrome with constipation and improvements in digestive and extra-digestive symptoms.

BACKGROUND: Linaclotide is approved for the treatment of moderate-to-severe irritable bowel syndrome (IBS) with constipation (IBS-C) in adults. This study aimed to assess factors predictive of a clinical response and improvements in non-IBS symptoms with linaclotide treatment in a Spanish patient population. METHODS: In this open-label phase IIIb study, patients with moderate-to-severe IBS-C received linaclotide 290 µg once daily for 12 weeks. The primary endpoint was clinical response at week 12, defined as >30% reduction in IBS symptom severity score (IBS-SSS) or IBS-SSS <75 plus self-reported response of feeling 'better' or 'much better' versus the baseline. Digestive nonintestinal and extra-digestive symptom scores were assessed. Baseline characteristics and week 4 clinical response were assessed as predictors of week 12 clinical response. RESULTS: A total of 96 patients were eligible; 91 were female and the mean age was 47.4 years. Mean (SD) baseline IBS-SSS was 371 (72.5). In the intention-to-treat and per-protocol populations, 22.9% and 31.7% were clinical responders at week 4, respectively, and 25.0% and 36.7% were clinical responders at week 12. Digestive nonintestinal and extra-digestive symptom scores were significantly improved at weeks 4 and 12. Baseline characteristic was not associated with week 12 clinical response; however, clinical response at week 4 was predictive of response at week 12 (OR: 6.5; 95%IC: 2.1-19.8). The most common adverse event was diarrhea inclusive of loose or watery stools (35.4%). CONCLUSIONS: Linaclotide improves IBS-C symptoms, including digestive nonintestinal and extra-digestive symptoms. A clinical response at week 4 may predict response at week 12.

Semaphorin-Plexin Signaling Controls Mitotic Spindle Orientation during Epithelial Morphogenesis and Repair.

Morphogenesis, homeostasis, and regeneration of epithelial tissues rely on the accurate orientation of cell divisions, which is specified by the mitotic spindle axis. To remain in the epithelial plane, symmetrically dividing epithelial cells align their mitotic spindle axis with the plane. Here, we show that this alignment depends on epithelial cell-cell communication via semaphorin-plexin signaling. During kidney morphogenesis and repair, renal tubular epithelial cells lacking the transmembrane receptor Plexin-B2 or its semaphorin ligands fail to correctly orient the mitotic spindle, leading to severe defects in epithelial architecture and function. Analyses of a series of transgenic and knockout mice indicate that Plexin-B2 controls the cell division axis by signaling through its GTPase-activating protein (GAP) domain and Cdc42. Our data uncover semaphorin-plexin signaling as a central regulatory mechanism of mitotic spindle orientation necessary for the alignment of epithelial cell divisions with the epithelial plane.

WIP modulates dendritic spine actin cytoskeleton by transcriptional control of lipid metabolic enzymes.

We identify Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP) as a novel component of neuronal synapses whose absence increases dendritic spine size and filamentous actin levels in an N-WASP/Arp2/3-independent, RhoA/ROCK/profilinIIa-dependent manner. These effects depend on the reduction of membrane sphingomyelin (SM) due to transcriptional upregulation of neutral sphingomyelinase (NSM) through active RhoA; this enhances RhoA binding to the membrane, raft partitioning and activation in steady state but prevents RhoA changes in response to stimulus. Inhibition of NSM or SM addition reverses RhoA, filamentous actin and functional anomalies in synapses lacking WIP. Our findings characterize WIP as a link between membrane lipid composition and actin cytoskeleton at dendritic spines. They also contribute to explain cognitive deficits shared by individuals bearing mutations in the region assigned to the gene encoding for WIP.

Integrin linked kinase (ILK) regulates podosome maturation and stability in dendritic cells.

Podosomes are integrin-based adhesions fundamental for stabilisation of the leading lamellae in migrating dendritic cells (DCs) and for extracellular matrix (ECM) degradation. We have previously shown that soluble factors and chemokines such as SDF 1-a trigger podosome initiation whereas integrin ligands promote podosome maturation and stability in DCs. The exact intracellular signalling pathways that regulate the sequential organisation of podosomal components in response to extracellular cues remain largely undetermined. The Wiskott Aldrich Syndrome Protein (WASP) mediates actin polymerisation and the initial recruitment of integrins and associated proteins in a circular configuration surrounding the core of filamentous actin (F-actin) during podosome initiation. We have now identified integrin linked kinase (ILK) surrounding the podosomal actin core. We report that DC polarisation in response to chemokines and the assembly of actin cores during podosome initiation require PI3K-dependent clustering of the Wiskott Aldrich Syndrome Protein (WASP) in puncta independently of ILK. ILK is essential for the clustering of integrins and associated proteins leading to podosome maturation and stability that are required for degradation of the subjacent extracellular matrix and the invasive motility of DCs across connective tissue barriers. We conclude that WASP regulates DCs polarisation for migration and initiation of actin polymerisation downstream of PI3K in nascent podosomes. Subsequently, ILK mediates the accumulation of integrin-associated proteins during podosome maturation and stability for efficient degradation of the subjacent ECM during the invasive migration of DCs.

EGFR controls IQGAP basolateral membrane localization and mitotic spindle orientation during epithelial morphogenesis.

Establishing the correct orientation of the mitotic spindle is an essential step in epithelial cell division in order to ensure that epithelial tubules form correctly during organ development and regeneration. While recent findings have identified some of the molecular mechanisms that underlie spindle orientation, many aspects of this process remain poorly understood. Here, we have used the 3D-MDCK model system to demonstrate a key role for a newly identified protein complex formed by IQGAP1 and the epithelial growth factor receptor (EGFR) in controlling the orientation of the mitotic spindle. IQGAP1 is a scaffolding protein that regulates many cellular pathways, from cell-cell adhesion to microtubule organization, and its localization in the basolateral membrane ensures correct spindle orientation. Through its IQ motifs, IQGAP1 binds to EGFR, which is responsible for maintaining IQGAP1 in the basolateral membrane domain. Silencing IQGAP1, or disrupting the basolateral localization of either IQGAP1 or EGFR, results in a non-polarized distribution of NuMA, mitotic spindle misorientation and defects in single lumen formation.

WIP regulates persistence of cell migration and ruffle formation in both mesenchymal and amoeboid modes of motility.

The spatial distribution of signals downstream from receptor tyrosine kinases (RTKs) or G-protein coupled receptors (GPCR) regulates fundamental cellular processes that control cell migration and growth. Both pathways rely significantly on actin cytoskeleton reorganization mediated by nucleation-promoting factors such as the WASP-(Wiskott-Aldrich Syndrome Protein) family. WIP (WASP Interacting Protein) is essential for the formation of a class of polarised actin microdomain, namely dorsal ruffles, downstream of the RTK for PDGF (platelet-derived growth factor) but the underlying mechanism is poorly understood. Using lentivirally-reconstituted WIP-deficient murine fibroblasts we define the requirement for WIP interaction with N-WASP (neural WASP) and Nck for efficient dorsal ruffle formation and of WIP-Nck binding for fibroblast chemotaxis towards PDGF-AA. The formation of both circular dorsal ruffles in PDGF-AA-stimulated primary fibroblasts and lamellipodia in CXCL13-treated B lymphocytes are also compromised by WIP-deficiency. We provide data to show that a WIP-Nck signalling complex interacts with RTK to promote polarised actin remodelling in fibroblasts and provide the first evidence for WIP involvement in the control of migratory persistence in both mesenchymal (fibroblast) and amoeboid (B lymphocytes) motility.

Synaptotagmin-like proteins control the formation of a single apical membrane domain in epithelial cells.

The formation of epithelial tissues requires both the generation of apical-basal polarity and the coordination of this polarity between neighbouring cells to form a central lumen. During de novo lumen formation, vectorial membrane transport contributes to the formation of a singular apical membrane, resulting in the contribution of each cell to only a single lumen. Here, from a functional screen for genes required for three-dimensional epithelial architecture, we identify key roles for synaptotagmin-like proteins 2-a and 4-a (Slp2-a/4-a) in the generation of a single apical surface per cell. Slp2-a localizes to the luminal membrane in a PtdIns(4,5)P(2)-dependent manner, where it targets Rab27-loaded vesicles to initiate a single lumen. Vesicle tethering and fusion is controlled by Slp4-a, in conjunction with Rab27/Rab3/Rab8 and the SNARE syntaxin-3. Together, Slp2-a/4-a coordinate the spatiotemporal organization of vectorial apical transport to ensure that only a single apical surface, and thus the formation of a single lumen, occurs per cell.

The cortactin-binding domain of WIP is essential for podosome formation and extracellular matrix degradation by murine dendritic cells.

In immature dendritic cells (DCs) podosomes form and turn over behind the leading edge of migrating cells. The Arp2/3 complex activator Wiskott-Aldrich Syndrome Protein (WASP) localises to the actin core of forming podosomes together with WASP-Interacting Protein (WIP). A second weaker Arp2/3 activator, cortactin, is also found at podosomes where it has been proposed to participate in matrix metalloproteinase (MMP) secretion. We have previously shown that WIP(-/-) DCs are unable to make podosomes. WIP binds to cortactin and in this report we address whether WIP regulates cortactin-mediated MMP activity. Using DCs derived from splenic murine precursors, we found that wild-type cells were able to localise MMPs at podosomes where matrix degradation takes place. In contrast, WIP(-/-) DCs remain able to synthesise MMPs but do not degrade the extracellular matrix. Infection of WIP KO DCs with lentivirus expressing WIP restored both podosome formation and their ability to degrade the extracellular matrix, implicating WIP-induced podosomes as foci of functional MMP location. When WIP KO DCs were infected with a mutant form of WIP lacking the cortactin-binding domain (WIPΔ110-170) DCs were only able to elaborate disorganised podosomes that were unable to support MMP-mediated matrix degradation. Taken together, these results suggest a role for WIP not only in WASP-mediated actin polymerisation and podosome formation, but also in cortactin-mediated extracellular matrix degradation by MMPs.

Role of WASP in cell polarity and podosome dynamics of myeloid cells.

The integrin-dependent migration of myeloid cells requires tight coordination between actin-based cell membrane protrusion and integrin-mediated adhesion to form a stable leading edge. Under this mode of migration, polarised myeloid cells including dendritic cells, macrophages and osteoclasts develop podosomes that sustain the extending leading edge. Podosome integrity and dynamics vary in response to changes in the physical and biochemical properties of the cell environment. In the current article we discuss the role of various factors in initiation and stability of podosomes and the roles of the Wiskott Aldrich Syndrome Protein (WASP) in this process. We discuss recent data indicating that in a cellular context WASP is crucial not only for localised actin polymerisation at the leading edge and in podosome cores but also for coordination of integrin clustering and activation during podosome formation and disassembly.

CD44 and beta3 integrin organize two functionally distinct actin-based domains in osteoclasts.

The actin cytoskeleton of mature osteoclasts (OCs) adhering to nonmineralized substrates is organized in a belt of podosomes reminiscent of the sealing zone (SZ) found in bone resorbing OCs. In this study, we demonstrate that the belt is composed of two functionally different actin-based domains: podosome cores linked with CD44, which are involved in cell adhesion, and a diffuse cloud associated with beta3 integrin, which is involved in cell adhesion and contraction. Wiskott Aldrich Syndrome Protein (WASp) Interacting Protein (WIP)-/- OCs were devoid of podosomes, but they still exhibited actin clouds. Indeed, WIP-/- OCs show diminished expression of WASp, which is required for podosome formation. CD44 is a novel marker of OC podosome cores and the first nonintegrin receptor detected in these structures. The importance of CD44 is revealed by showing that its clustering restores podosome cores and WASp expression in WIP-/- OCs. However, although CD44 signals are sufficient to form a SZ, the presence of WIP is indispensable for the formation of a fully functional SZ.

Expression of cadherins and catenins correlates with distinct histologic types of ovarian carcinomas.

Alterations in the cadherin-catenin expression and activation of the Wnt signaling have been related to the pathology of ovarian carcinomas. Here, we evaluated the immunoreactivity of cadherins (E-, P-, and N-cadherin and cadherin-11) and catenins (alpha-, beta-, and gamma-catenin and p120) in 86 ovarian tumors. We found significant differences in the expression of all cadherins and catenins among the distinct histologic tumor types. Clear cell tumors were rarely N-cadherin- and P-cadherin-positive and showed reduced membranous expression in all the catenins; Serous carcinomas were frequently N-cadherin- and P-cadherin-positive, mucinous tumors strongly expressed E-cadherin and the catenins in the membrane, and endometrioid tumors characteristically expressed nucleocytoplasmic beta-catenin in most of the cases. We next studied whether allelic losses in the chromosomal regions containing various cadherin genes (16q22) or APC gene (5q21) occurred in ovarian tumors and observed a high frequency of loss of heterozygosity in 16q22 (78%) and 5q21 (33%) regions, but there were no differences among the tumor types analyzed. Finally, we also assessed the molecular alterations responsible for beta-catenin nuclear accumulation in endometrioid tumors by screening for mutations in AXIN1, AXIN2, APC, and KRAS genes. Mutations in KRAS were observed in 2 of 19 tumors, but no mutations were detected in AXIN1, AXIN2, or APC genes. Only beta-catenin gene mutations were associated with nuclear beta-catenin staining in these tumors. In conclusion, different cadherin-catenin expression patterns are associated with distinct histologic types. Oncogenic Wnt signaling plays a role only in endometrioid tumors, where beta-catenin mutations seem to be the main cause of its aberrant expression.

Transcriptional profiling of MCF7 breast cancer cells in response to 5-Fluorouracil: relationship with cell cycle changes and apoptosis, and identification of novel targets of p53.

The availability of oral precursors of 5-Fluorouracil (5-FU) and its favorable results in treating advanced breast cancer have renewed the interest in the molecular mechanisms underlying its cytotoxicity. We have compared the changes in cell cycle and cell death parameters induced by 2 different concentrations of 5-FU (IC50 and IC80) in the breast adenocarcinoma cell line MCF7. G1/S cell cycle arrest was associated with both concentrations, whereas cell death was mainly induced after IC80 5-FU. These changes were correlated with gene expression assessed by cDNA microarray analysis. Main findings included an overexpression of p53 target genes involved in cell cycle and apoptosis (CDKN1A/p21, TP53INP, TNFRSF6/FAS and BBC3/PUMA), and significant repression of Myc. High dose 5-FU also induced a higher regulation of the mitochondrial death genes APAF1, BAK1 and BCL2, and induction of genes of the ID family. Furthermore, we establish a direct causal relationship between p21, ID1 and ID2 overexpression, increased acetylation of histones H3 and H4 and binding of p53 to their promoters as a result of 5-FU treatment. The relevance of these findings was further studied after interfering p53 expression in MCF7 cells (shp53 cells), showing a lower induction of both, ID1 and ID2 transcripts, after 5-FU when compared with MCF7 shGFP control cells. This molecular characterization of dose- and time-dependent modifications of gene expression after 5-FU treatment should provide a resource for future basic studies addressing the molecular mechanisms of chemotherapy in breast cancer.