Genetic coding variant in complement factor B (CFB) is associated with increased risk for perianal Crohn's disease and leads to impaired CFB cleavage and phagocytosis.

OBJECTIVE: Perianal Crohn's disease (pCD) occurs in up to 40% of patients with CD and is associated with poor quality of life, limited treatment responses and poorly understood aetiology. We performed a genetic association study comparing CD subjects with and without perianal disease and subsequently performed functional follow-up studies for a pCD associated SNP in Complement Factor B (CFB). DESIGN: Immunochip-based meta-analysis on 4056 pCD and 11 088 patients with CD from three independent cohorts was performed. Serological and clinical variables were analysed by regression analyses. Risk allele of rs4151651 was introduced into human CFB plasmid by site-directed mutagenesis. Binding of recombinant G252 or S252 CFB to C3b and its cleavage was determined in cell-free assays. Macrophage phagocytosis in presence of recombinant CFB or serum from CFB risk, or protective CD or healthy subjects was assessed by flow cytometry. RESULTS: Perianal complications were associated with colonic involvement, OmpC and ASCA serology, and serology quartile sum score. We identified a genetic association for pCD (rs4151651), a non-synonymous SNP (G252S) in CFB, in all three cohorts. Recombinant S252 CFB had reduced binding to C3b, its cleavage was impaired, and complement-driven phagocytosis and cytokine secretion were reduced compared with G252 CFB. Serine 252 generates a de novo glycosylation site in CFB. Serum from homozygous risk patients displayed significantly decreased macrophage phagocytosis compared with non-risk serum. CONCLUSION: pCD-associated rs4151651 in CFB is a loss-of-function mutation that impairs its cleavage, activation of alternative complement pathway, and pathogen phagocytosis thus implicating the alternative complement pathway and CFB in pCD aetiology.

Increased efficacy of dual proinflammatory cytokine blockade on acute GVHD while maintaining GVT effects.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains a potential curative option for treating a variety of hematologic diseases, but acute and chronic graft-versus-host disease (GVHD) remain major barriers limiting efficacy. Acute gut GVHD occurs with marked increases in proinflammatory cytokines (including TNF and IL-6), which we recently demonstrated was exacerbated in obesity resulting in severe gastrointestinal pathology. Given the pleiotropic and overlapping effects of these 2 cytokines, we assessed the impact of dual TNF and IL-6R blockade on GVHD as well as graft-versus tumor (GVT) effects in different mouse GVHD models. Early administration of combined blockade resulted in greater protection and survival from acute gut GVHD compared with single blockade regimens and even development of later chronic skin GVHD. Importantly, double cytokine blockade preserved GVT effects reinforcing that GVT and GVHD can be delineated and may result in greater efficacy in allo-HSCT.

Schlafen 12 Interaction with SerpinB12 and Deubiquitylases Drives Human Enterocyte Differentiation.

BACKGROUND/AIMS: Human enterocytic differentiation is altered during development, fasting, adaptation, and bariatric surgery, but its intracellular control remains unclear. We hypothesized that Schlafen 12 (SLFN12) regulates enterocyte differentiation. METHODS: We used laser capture dissection of epithelium, qRT-PCR, and immunohistochemistry to evaluate SLFN12 expression in biopsies of control and fasting human duodenal mucosa, and viral overexpression and siRNA to trace the SLFN12 pathway in human Caco-2 and HIEC6 intestinal epithelial cells. RESULTS: Fasting human duodenal mucosa expressed less SLFN12 mRNA and protein, accompanied by decreases in enterocytic markers like sucrase-isomaltase. SLFN12 overexpression increased Caco-2 sucrase-isomaltase promoter activity, mRNA, and protein independently of proliferation, and activated the SLFN12 putative promoter. SLFN12 coprecipitated Serpin B12 (SERPB12). An inactivating SLFN12 point mutation prevented both SERPB12 binding and sucrase-isomaltase induction. SERPB12 overexpression also induced sucrase-isomaltase, while reducing SERPB12 prevented the SLFN12 effect on sucrase-isomaltase. Sucrase-isomaltase induction by both SLFN12 and SERPB12 was attenuated by reducing UCHL5 or USP14, and blocked by reducing both. SERPB12 stimulated USP14 but not UCHL5 activity. SERPB12 coprecipitated USP14 but not UCHL5. Moreover, SLFN12 increased protein levels of the sucrase-isomaltase-promoter-binding transcription factor cdx2 without altering Cdx2 mRNA. This was prevented by reducing UCHL5 and USP14. We further validated this pathway in vitro and in vivo. SLFN12 or SERPB12 overexpression induced sucrase-isomaltase in human non-malignant HIEC-6 enterocytes. CONCLUSIONS: SLFN12 regulates human enterocytic differentiation by a pathway involving SERPB12, the deubiquitylases, and Cdx2. This pathway may be targeted to manipulate human enterocytic differentiation in mucosal atrophy, short gut or obesity.

Galectin-3-induced cell spreading and motility relies on distinct signaling mechanisms compared to fibronectin.

Secreted galectin-3 often gets incorporated into extracellular matrix and is utilized by cancer cells for spreading, movement, and metastatic dissemination. Here we investigate molecular mechanisms by which galectin-3 brings about these effects and compare it with fibronectin. Imaging of cells spread on fibronectin showed stress fibers throughout cell body, however, galectin-3-induced formation of parallel actin bundles in the lamellipodial region resulting in unique morphological features. FRAP analysis showed that the actin turnover in the lamellipodial region was much higher in cells spread on galectin-3 as compared to that on fibronectin. Rac1 activation is correlated with lamellipodial organization on both the substrates. Activation of Akt and Rac1, the regulators of actin dynamics, show inverse correlation with each other on both galectin-3 and fibronectin. Activation of Erk however, remained similar. Further, inhibition of activation of Akt and Erk inhibited spreading and motility of cells on galectin-3 but not on fibronectin. The results very comprehensively demonstrate distinct signaling pathways that regulate microfilament organization, lamellipodial structures, spreading, and movement of cells plated on galectin-3 as opposed to fibronectin.

N-glycans and metastasis in galectin-3 transgenic mice.

Poly-N-acetyl-lactosamine (polyLacNAc) on N-glycans facilitate lung specific metastasis of melanoma cells by serving as high affinity ligands for galectin-3, expressed in highest amounts in the lungs, on almost all its tissue compartments including on the surface of vascular endothelium. PolyLacNAc not only aids in initial arrest on the organ endothelium but in all the events of extravasation. Inhibition of polyLacNAc synthesis, or competitive inhibition of its interaction with galectin-3 all inhibited these processes and experimental metastasis. Transgenic galectin-3 mice, viz., gal-3(+/+) (wild type), gal-3(+/-) (hemizygous) and gal-3(-/-) (null) have been used to prove that galectin-3/polyLacNAc interactions are indeed critical for lung specific metastasis. Gal-3(+/-) mice which showed <50% expression of galectin-3 on the lungs also showed proportionate decrease in the number of B16F10 melanoma metastatic colonies affirming that galectin-3 and polyLacNAc interactions are indeed key determinants of lung metastasis. However, surprisingly, the number and size of metastatic colonies in gal-3(-/-) mice was very similar as that seen in gal-3(+/+) mice. The levels of lactose binding lectins on the lungs and the transcripts of other galectins (galectin-1, -8 and -9) which are expressed on lungs and have similar sugar binding specificities as galectins-3, remain unchanged in gal-3(+/+) and gal-3(-/-) mice. Further, inhibition of N-glycosylation with Swainsonine (SW) which drastically reduces metastasis of B16F10 cells in gal-3(+/+) mice, did not affect lung metastasis when assessed in gal-3(-/-) mice. Together, these results rule out the possibility of some other galectin taking over the function of galectin-3 in gal-3(-/-) mice. Chimeric mice generated to assess if absence of any effect on metastasis is due to compromised tumor immunity by replacing bone marrow of gal-3(-/-) mice with that from gal-3(+/+) mice, also failed to impact melanoma metastasis. As galectin-3 regulates several immune functions including maturation of different immune cells, compromised tumor immunity could be the major determinant of melanoma metastasis in gal-3(-/-) mice and warrants thorough investigation.

The role of intestine in metabolic dysregulation in murine Wilson disease.

Background and aims: Major clinical manifestations of Wilson disease (WD) are related to copper accumulation in the liver and the brain, and little is known about other tissues involvement in metabolic changes in WD. In vitro studies suggested that the loss of intestinal ATP7B could contribute to metabolic dysregulation in WD. We tested this hypothesis by evaluating gut microbiota and lipidome in two mouse models of WD and by characterizing a new mouse model with a targeted deletion of Atp7b in intestine. Methods: Cecal content 16S sequencing and untargeted hepatic and plasma lipidome analyses in the Jackson Laboratory toxic-milk and the Atp7b null global knockout mouse models of WD were profiled and integrated. Intestine-specific Atp7b knockout mice ( Atp7b ΔIEC ) was generated using B6.Cg-Tg(Vil1-cre)997Gum/J mice and Atp7b Lox/Lox mice, and characterized using targeted lipidome analysis following a high-fat diet challenge. Results: Gut microbiota diversity was reduced in animal models of WD. Comparative prediction analysis revealed amino acid, carbohydrate, and lipid metabolism functions to be dysregulated in the WD gut microbial metagenome. Liver and plasma lipidomic profiles showed dysregulated tri- and diglyceride, phospholipid, and sphingolipid metabolism in WD models. When challenged with a high-fat diet, Atp7b ΔIEC mice exhibited profound alterations to fatty acid desaturation and sphingolipid metabolism pathways as well as altered APOB48 distribution in intestinal epithelial cells. Conclusion: Coordinated changes of gut microbiome and lipidome analyses underlie systemic metabolic manifestations in murine WD. Intestine-specific ATP7B deficiency affected both intestinal and systemic response to a high-fat challenge. WD is a systemic disease in which intestinal-specific ATP7B loss and diet influence phenotypic presentations.

DR3 Regulates Intestinal Epithelial Homeostasis and Regeneration After Intestinal Barrier Injury.

BACKGROUND & AIMS: Tumor necrosis factor (TNF) superfamily member tumor necrosis factor-like protein 1A (TL1A) has been associated with the susceptibility and severity of inflammatory bowel diseases. However, the function of the tumor necrosis factor-like protein 1A and its receptor death receptor 3 (DR3) in the development of intestinal inflammation is incompletely understood. We investigated the role of DR3 expressed by intestinal epithelial cells (IECs) during intestinal homeostasis, tissue injury, and regeneration. METHODS: Clinical phenotype and histologic inflammation were assessed in C57BL/6 (wild-type), Tl1a-/- and Dr3-/- mice in dextran sulfate sodium (DSS)-induced colitis. We generated mice with an IEC-specific deletion of DR3 (Dr3ΔIEC) and assessed intestinal inflammation and epithelial barrier repair. In vivo intestinal permeability was assessed by fluorescein isothiocyanate dextran uptake. Proliferation of IECs was analyzed by bromodeoxyuridine incorporation. Expression of DR3 messenger RNA was assessed by fluorescent in situ hybridization. Small intestinal organoids were used to determine ex vivo regenerative potential. RESULTS: Dr3-/- mice developed more severe colonic inflammation than wild-type mice in DSS-induced colitis with significantly impaired IEC regeneration. Homeostatic proliferation of IECs was increased in Dr3-/- mice, but blunted during regeneration. Cellular localization and expression of the tight junction proteins Claudin-1 and zonula occludens-1 were altered, leading to increased homeostatic intestinal permeability. Dr3ΔIEC mice recapitulated the phenotype observed in Dr3-/- mice with increased intestinal permeability and IEC proliferation under homeostatic conditions and impaired tissue repair and increased bacterial translocation during DSS-induced colitis. Impaired regenerative potential and altered zonula occludens-1 localization also were observed in Dr3ΔIEC enteroids. CONCLUSIONS: Our findings establish a novel function of DR3 in IEC homeostasis and postinjury regeneration independent of its established role in innate lymphoid cells and T-helper cells.

The role of intestine in metabolic dysregulation in murine Wilson disease.

BACKGROUND: The clinical manifestations of Wilson disease (WD) are related to copper accumulation in the liver and the brain, but little is known about other tissue involvement regarding metabolic changes in WD. In vitro studies suggested that the loss of intestinal ATP7B affects metabolic dysregulation in WD. We tested this hypothesis by evaluating the gut microbiota and lipidome in 2 mouse models of WD and by characterizing a new mouse model with a targeted deletion of Atp7b in the intestine. METHODS: Cecal content 16S sequencing and untargeted hepatic and plasma lipidome analyses in the Jackson Laboratory toxic-milk and the Atp7b null global knockout mouse models of WD were profiled and integrated. Intestine-specific Atp7b knockout mice (Atp7bΔIEC) were generated and characterized using targeted lipidome analysis following a high-fat diet challenge. RESULTS: Gut microbiota diversity was reduced in animal models of WD. Comparative prediction analysis revealed amino acid, carbohydrate, and lipid metabolism functions to be dysregulated in the WD gut microbial metagenome. Liver and plasma lipidomic profiles showed dysregulated triglyceride and diglyceride, phospholipid, and sphingolipid metabolism in WD models. However, Atp7bΔIEC mice did not show gut microbiome differences compared to wild type. When challenged with a high-fat diet, Atp7bΔIEC mice exhibited profound alterations to fatty acid desaturation and sphingolipid metabolism pathways as well as altered APOB48 distribution in intestinal epithelial cells. CONCLUSIONS: Gut microbiome and lipidome underlie systemic metabolic manifestations in murine WD. Intestine-specific ATP7B deficiency affected both intestinal and systemic response to a high-fat challenge but not the microbiome profile, at least at early stages. WD is a systemic disease in which intestinal-specific ATP7B loss and diet influence the phenotype and the lipidome profile.

Biomarker Potential of Vimentin in Oral Cancers.

Oral carcinogenesis is a multistep process. As much as 5% to 85% of oral tumors can develop from potentially malignant disorders (PMD). Although the oral cavity is accessible for visual examination, the ability of current clinical or histological methods to predict the lesions that can progress to malignancy is limited. Thus, developing biological markers that will serve as an adjunct to histodiagnosis has become essential. Our previous studies comprehensively demonstrated that aberrant vimentin expression in oral premalignant lesions correlates to the degree of malignancy. Likewise, overwhelming research from various groups show a substantial contribution of vimentin in oral cancer progression. In this review, we have described studies on vimentin in oral cancers, to make a compelling case for vimentin as a prognostic biomarker.

BATF3 Protects Against Metabolic Syndrome and Maintains Intestinal Epithelial Homeostasis.

The intestinal immune system and microbiota are emerging as important contributors to the development of metabolic syndrome, but the role of intestinal dendritic cells (DCs) in this context is incompletely understood. BATF3 is a transcription factor essential in the development of mucosal conventional DCs type 1 (cDC1). We show that Batf3-/- mice developed metabolic syndrome and have altered localization of tight junction proteins in intestinal epithelial cells leading to increased intestinal permeability. Treatment with the glycolysis inhibitor 2-deoxy-D-glucose reduced intestinal inflammation and restored barrier function in obese Batf3-/- mice. High-fat diet further enhanced the metabolic phenotype and susceptibility to dextran sulfate sodium colitis in Batf3-/- mice. Antibiotic treatment of Batf3-/- mice prevented metabolic syndrome and impaired intestinal barrier function. Batf3-/- mice have altered IgA-coating of fecal bacteria and displayed microbial dysbiosis marked by decreased obesity protective Akkermansia muciniphila, and Bifidobacterium. Thus, BATF3 protects against metabolic syndrome and preserves intestinal epithelial barrier by maintaining beneficial microbiota.

Efficacy of Cilnidipine (L/N-type Calcium Channel Blocker) in Treatment of Hypertension: A Meta-Analysis of Randomized and Non-randomized Controlled Trials.

INTRODUCTION:  Hypertension is one of the most common cardiovascular diseases, and the prevalence of hypertension continues to rise across the globe. National and international guidelines recommend angiotensin-converting enzyme (ACE) inhibitors or angiotensin receptor blockers (ARBs), calcium channel blockers (CCBs), diuretics, and beta-blockers for the management of hypertension. CCBs are among the most used antihypertensive medications and Cilnidipine is a newer dihydropyridine CCB shown to have a prolonged antihypertensive property. OBJECTIVE:  This meta-analysis of comparative randomized and non-randomized clinical trials evaluated the effect of Cilnidipine monotherapy or combination therapy on systolic blood pressure (SBP), diastolic blood pressure (DBP), and pulse rate (PR) over 48 weeks of therapy. STUDY DESIGN:  PubMed (MEDLINE) and Google scholar databases were searched to identify studies designed to evaluate the effects of Cilnidipine in the treatment of hypertensive patients. The study criteria for inclusion into the meta-analysis were all prospective, randomized, and non-randomized clinical studies published till March 2021, studies published in a peer-reviewed journal, the inclusion of patients with hypertension, assessment of blood pressure and heart rate, and a follow-up of four weeks or longer. The initial search identified 82 potential articles; of these, 24 met the inclusion criteria. Studies with <4 weeks treatment period and those not having a CCB were excluded. OUTCOMES:  Change in SBP, DBP, and PR from baseline at the end of therapy compared between the Cilnidipine and other CCB's. RESULTS: Cilnidipine caused a significant reduction (p<0.05) in SBP, DBP, and PR at end of therapy, whereas the reduction in SBP, DBP, and PR with Cilnidipine was similar to other CCB's (p>0.05). The results of this meta-analysis revealed that there were no significant differences in the efficacy in the treatment of hypertensive patients with Cilnidipine and the other therapies. CONCLUSION:  Cilnidipine has similar anti-hypertensive effects compared with other first-line antihypertensive drugs commonly used in practice. We recommend Cilnidipine as a novel first-line CCB for the management of hypertension either as a monotherapy or as a combination therapy.

ZINC40099027 activates human focal adhesion kinase by accelerating the enzymatic activity of the FAK kinase domain.

Focal adhesion kinase (FAK) regulates gastrointestinal epithelial restitution and healing. ZINC40099027 (Zn27) activates cellular FAK and promotes intestinal epithelial wound closure in vitro and in mice. However, whether Zn27 activates FAK directly or indirectly remains unknown. We evaluated Zn27 potential modulation of the key phosphatases, PTP-PEST, PTP1B, and SHP2, that inactivate FAK, and performed in vitro kinase assays with purified FAK to assess direct Zn27-FAK interaction. In human Caco-2 cells, Zn27-stimulated FAK-Tyr-397 phosphorylation despite PTP-PEST inhibition and did not affect PTP1B-FAK interaction or SHP2 activity. Conversely, in vitro kinase assays demonstrated that Zn27 directly activates both full-length 125 kDa FAK and its 35 kDa kinase domain. The ATP-competitive FAK inhibitor PF573228 reduced basal and ZN27-stimulated FAK phosphorylation in Caco-2 cells, but Zn27 increased FAK phosphorylation even in cells treated with PF573228. Increasing PF573228 concentrations completely prevented activation of 35 kDa FAK in vitro by a normally effective Zn27 concentration. Conversely, increasing Zn27 concentrations dose-dependently activated kinase activity and overcame PF573228 inhibition of FAK, suggesting the direct interactions of Zn27 with FAK may be competitive. Zn27 increased the maximal activity (Vmax ) of FAK. The apparent Km of the substrate also increased under laboratory conditions less relevant to intracellular ATP concentrations. These results suggest that Zn27 is highly potent and enhances FAK activity via allosteric interaction with the FAK kinase domain to increase the Vmax of FAK for ATP. Understanding Zn27 enhancement of FAK activity will be important to redesign and develop a clinical drug that can promote mucosal wound healing.

Discovery of Novel Small-Molecule FAK Activators Promoting Mucosal Healing.

Gastrointestinal mucosal wounds are common to patients injured by factors as diverse as drugs, inflammatory bowel disease, peptic ulcers, and necrotizing enterocolitis. However, although many drugs are used to ameliorate injurious factors, there is no drug available to actually stimulate mucosal wound healing. Focal adhesion kinase (FAK), a nonreceptor tyrosine kinase, induces epithelial sheet migration and wound healing, making FAK a potential pharmacological target in this regard. In our previous research, we found a lead compound with drug-like properties, ZINC40099027, which promotes FAK phosphorylation, inducing mucosal healing in murine models. Herein we describe the design and optimization of a small library of novel FAK activators based on ZINC40099027 and their applications toward human intestinal epithelial wound closure and mouse ulcer healing.

Intestinal Stem Cell Niche Defects Result in Impaired 3D Organoid Formation in Mouse Models of Crohn's Disease-like Ileitis.

Intestinal epithelial barrier dysfunction is a risk factor in the pathogenesis of Crohn's disease (CD); however, no corrective FDA-approved therapies exist. We used an enteroid (EnO)-based system in two murine models of experimental CD, SAMP1/YitFc (SAMP) and TNFΔARE/+ (TNF). While severely inflamed SAMP mice do not generate EnOs, "inflammation-free" SAMP mice form EnO structures with impaired morphology and reduced intestinal stem cell (ISC) and Paneth cell viability. We validated these findings in TNF mice concluding that inflammation in intestinal tissues impedes EnO generation and suppressing inflammation by steroid administration partially rescues impaired formation in SAMP mice. We generated the first high-resolution transcriptional profile of the SAMP ISC niche demonstrating that alterations in multiple key pathways contribute to niche defect and targeting them may partially rescue the phenotype. Furthermore, we correlated the defects in formation and the rescue of EnO formation to reduced viability of ISCs and Paneth cells.

Obesity induces gut microbiota alterations and augments acute graft-versus-host disease after allogeneic stem cell transplantation.

The efficacy of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is limited by acute and chronic graft-versus-host disease (GVHD). The impact of obesity on allo-HSCT outcomes is poorly understood. Here, we report that obesity had a negative and selective impact on acute gut GVHD after allo-HSCT in mice with diet-induced obesity (DIO). These animals exhibited increased gut permeability, endotoxin translocation across the gut, and radiation-induced gastrointestinal damage after allo-HSCT. After allo-HSCT, both male and female DIO mouse recipients showed increased proinflammatory cytokine production and expression of the GVHD marker ST2 (IL-33R) and MHC class II molecules; they also exhibited decreased survival associated with acute severe gut GVHD. This rapid-onset, obesity-associated gut GVHD depended on donor CD4+ T cells and occurred even with a minor MHC mismatch between donor and recipient animals. Retrospective analysis of clinical cohorts receiving allo-HSCT transplants from unrelated donors revealed that recipients with a high body mass index (BMI, >30) had reduced survival and higher serum ST2 concentrations compared with nonobese transplant recipients. Assessment of both DIO mice and allo-HSCT recipients with a high BMI revealed reduced gut microbiota diversity and decreased Clostridiaceae abundance. Prophylactic antibiotic treatment protected DIO mouse recipients from endotoxin translocation across the gut and increased inflammatory cytokine production, as well as gut pathology and mortality, but did not protect against later development of chronic skin GVHD. These results suggest that obesity-induced alterations of the gut microbiota may affect GVHD after allo-HSCT in DIO mice, which could be ameliorated by prophylactic antibiotic treatment.

ZINC4085554 inhibits cancer cell adhesion by interfering with the interaction of Akt1 and FAK.

Perioperative or circulatory forces enhance disseminated cancer cell adhesiveness by modulating focal adhesion kinase (FAK)-Akt1 interaction. Selectively blocking FAK-Akt1 interaction by a peptide derived from the FAK-Four-point-one, ezrin, radixin, moesin (FERM) domain reduces colon cancer cell adhesion in vitro and in mice. A preliminary in silico screening identified two small molecules resembling a peptide that may inhibit pressure-stimulated SW620 cancer cell adhesion to collagen I. The present study selected ZINC4085554 for further study to validate its proposed mechanism of action, using human SW620 colon cancer cells as a model system. At 25 and 50 µM, ZINC4085554 inhibited the pressure-stimulated adhesion of SW620 colon cancer cells to collagen I. This molecule prevented pressure-stimulated FAK-Tyr-397 phosphorylation; however, it did not affect Akt1-Ser-473 phosphorylation, indicating that ZINC4085554 acts downstream of Akt1, while Akt-Thr-308 remains unchanged in the presence of pressure and or ZINC4085554. Indeed, ZINC4085554 inhibited FAK-Akt1 interaction in response to increased extracellular pressure, consistent with the proposed mechanism. ZINC4085554 did not inhibit FAK-Tyr-397 phosphorylation in response to cell adhesion to collagen I, indicating the specificity of the inhibitory effects towards force-stimulated pathways. Finally, the present study confirmed that ZINC4085554 at 50 µM prevented pressure-activation of adhesion to surgical wounds in vivo in parallel to its ablation of intracellular signaling. In summary, ZINC4085554 is a small molecule mimicking part of the structure of FAK that reduces cancer cell adhesion by impairing pressure-stimulated FAK-Akt1 interaction and its downstream consequences. ZINC4085554 does not inhibit conventional outside-in FAK signaling and may be less toxic than global FAK inhibitors, and ZINC4085554 may be an important step towards the inhibition of metastasis.

Small molecule FAK activator promotes human intestinal epithelial monolayer wound closure and mouse ulcer healing.

GI mucosal healing requires epithelial sheet migration. The non-receptor tyrosine kinase focal adhesion kinase (FAK) stimulates epithelial motility. A virtual screen identified the small drug-like FAK mimic ZINC40099027, which activates FAK. We assessed whether ZINC40099027 promotes FAK-Tyr-397 phosphorylation and wound healing in Caco-2 monolayers and two mouse intestinal injury models. Murine small bowel ulcers were generated by topical serosal acetic acid or subcutaneous indomethacin in C57BL/6J mice. One day later, we began treatment with ZINC40099027 or DMSO, staining the mucosa for phosphorylated FAK and Ki-67 and measuring mucosal ulcer area, serum creatinine, ALT, and body weight at day 4. ZINC40099027 (10-1000 nM) dose-dependently activated FAK phosphorylation, without activating Pyk2-Tyr-402 or Src-Tyr-419. ZINC40099027 did not stimulate proliferation, and stimulated wound closure independently of proliferation. The FAK inhibitor PF-573228 prevented ZINC40099027-stimulated wound closure. In both mouse ulcer models, ZINC40099027accelerated mucosal wound healing. FAK phosphorylation was increased in jejunal epithelium at the ulcer edge, and Ki-67 staining was unchanged in jejunal mucosa. ZINC40099027 serum concentration at sacrifice resembled the effective concentration in vitro. Weight, creatinine and ALT did not differ between groups. Small molecule FAK activators can specifically promote epithelial restitution and mucosal healing and may be useful to treat gut mucosal injury.

Schlafen 12 mediates the effects of butyrate and repetitive mechanical deformation on intestinal epithelial differentiation in human Caco-2 intestinal epithelial cells.

Intestinal epithelial differentiation may be stimulated by diverse pathways including luminal short-chain fatty acids and repetitive mechanical deformation engendered by villous motility and peristalsis. Schlafen 12 (SLFN12) is a cytosolic protein that stimulates sucrase-isomaltase (SI) expression. We hypothesized that two disparate differentiating stimuli, butyrate and repetitive deformation, would each stimulate SLFN12 expression in human Caco-2 intestinal epithelial cells and that increased SLFN12 expression would contribute to the differentiating activity of the human Caco-2 intestinal epithelial cells. We stimulated Caco-2 cells with 1-2 mM butyrate or repetitive mechanical deformation at 10 cycles/min at an average 10% strain, and measured SLFN12 and SI expression by qRT-PCR. Sodium butyrate enhanced SLFN12 expression at both 1 mM and 2 mM although SI expression was only significantly increased at 2 mM. Repetitive deformation induced by cyclic mechanical strain also significantly increased both SLFN12 and SI gene expression. Reducing SLFN12 by siRNA decreased basal, deformation-stimulated, and butyrate-stimulated SLFN12 levels, compared to control cells treated with non-targeting siRNA, although both deformation and butyrate were still able to stimulate SLFN12 expression in siRNA-treated cells compared to control cells treated with the same siRNA. This attenuation of the increase in SLFN12 expression in response to mechanical strain or butyrate was accompanied by parallel attenuation of SI expression. Butyrate stimulated SI-promoter activity, and reducing SLFN12 by siRNA attenuated butyrate-induced SI-promoter activity. These data suggest that SLFN12 mediates at least in part the stimulation by both butyrate and repetitive mechanical deformation of sucrase-isomaltase, a late stage differentiation marker in human intestinal epithelial cells.

Mass spectrometry based identification of galectin-3 interacting proteins potentially involved in lung melanoma metastasis.

Adhesive interactions between molecules on tumor cells and those on target organs play a key role in organ specific metastasis. Poly-N-acetyl-lactosamine (polyLacNAc) substituted N-oligosaccharides on melanoma cell surface glycoproteins promote lung specific metastasis via galectin-3 by facilitating their arrest and extravasation. This study reports the identification and characterization of galectin-3 interacting proteins using a combination of galectin-3 sepharose affinity and leucoagglutinating phytohemagglutinin (L-PHA) columns. A total of 83 proteins were identified as galectin-3 interacting glycoproteins, of which 35 were constituents of the L-PHA bound fraction, suggesting that these proteins carry polyLacNAc substituted ß1,6 branched N-glycans. The identities of some of these proteins, like LAMP-1, LAMP-3, basigin, embigin, and α5 and ß1 Integrin, have been confirmed by western blotting, and functional relevance with respect to metastatic properties has been established.