Tetrapeptides Modelled to the Androgen Binding Site of ZIP9 Stimulate Expression of Tight Junction Proteins and Tight Junction Formation in Sertoli Cells

Androgens stimulate the expression of tight junction (TJ) proteins and the formation of the blood–testis barrier (BTB). Interactions of testosterone with the zinc transporter ZIP9 stimulate the expression of TJ-forming proteins and promote TJ formation in Sertoli cells. In order to investigate androgenic effects mediated by ZIP9 but not by the nuclear androgen receptor (AR), the effects of three tetrapeptides fitting the androgen binding site of ZIP9 were compared with those induced by testosterone in a Sertoli cell line expressing ZIP9 but not the AR. Three tetrapeptides and testosterone displaced testosterone-BSA-FITC from the surface of 93RS2 cells and stimulated the non-classical testosterone signaling pathway that includes the activation of Erk1/2 kinases and transcription factors CREB and ATF-1. The expression of the TJ-associated proteins ZO-1 and claudin-5 was triggered as was the re-distribution of claudin-1 from the cytosol to the membrane and nucleus. Furthermore, TJ formation was stimulated, indicated by increased transepithelial electrical resistance. Silencing ZIP9 expression by siRNA prevented all of these responses. These results are consistent with an alternative pathway for testosterone action at the BTB that does not involve the nuclear AR and highlight the significant role of ZIP9 as a cell-surface androgen receptor that stimulates TJ formation.

The NOTCH3 Downstream Target HEYL Is Required for Efficient Human Airway Basal Cell Differentiation

Basal cells (BCs) are stem/progenitor cells of the mucociliary airway epithelium, and their differentiation is orchestrated by the NOTCH signaling pathway. NOTCH3 receptor signaling regulates BC to club cell differentiation; however, the downstream responses that regulate this process are unknown. Overexpression of the active NOTCH3 intracellular domain (NICD3) in primary human bronchial epithelial cells (HBECs) on in vitro air–liquid interface culture promoted club cell differentiation. Bulk RNA-seq analysis identified 692 NICD3-responsive genes, including the classical NOTCH target HEYL, which increased in response to NICD3 and positively correlated with SCGB1A1 (club cell marker) expression. siRNA knockdown of HEYL decreased tight junction formation and cell proliferation. Further, HEYL knockdown reduced club, goblet and ciliated cell differentiation. In addition, we observed decreased expression of HEYL in HBECs from donors with chronic obstructive pulmonary disease (COPD) vs. normal donors which correlates with the impaired differentiation capacity of COPD cells. Finally, overexpression of HEYL in COPD HBECs promoted differentiation into club, goblet and ciliated cells, suggesting the impaired capacity of COPD cells to generate a normal airway epithelium is a reversible phenotype that can be regulated by HEYL. Overall, our data identify the NOTCH3 downstream target HEYL as a key regulator of airway epithelial differentiation.

A Chimeric Peptide Inhibits Red Blood Cell Invasion by Plasmodium falciparum with Hundredfold Increased Efficacy

Inhibition of tight junction formation between two malaria parasite proteins, apical membrane antigen 1 and rhoptry neck protein 2, crucial for red blood cell invasion, prevents the disease progression. In this work, we have utilized a unique approach to design a chimeric peptide, prepared by fusion of the best features of two peptide inhibitors, that has displayed parasite growth inhibition, in-vitro, with nanomolar IC50, which is hundredfold better than any of its parent peptides. Further, to gain structural insights, we computationally modeled the hybrid peptide on its receptor.

The F11 Receptor (F11R)/Junctional Adhesion Molecule-A (JAM-A) (F11R/JAM-A) in cancer progression

The F11 Receptor (F11R), also called Junctional Adhesion Molecule-A (JAM-A) (F11R/JAM-A), is a transmembrane glycoprotein of the immunoglobulin superfamily, which is mainly located in epithelial and endothelial cell tight junctions and also expressed on circulating platelets and leukocytes. It participates in the regulation of various biological processes, as diverse as paracellular permeability, tight junction formation and maintenance, leukocyte transendothelial migration, epithelial-to-mesenchymal transition, angiogenesis, reovirus binding, and platelet activation. Dysregulation of F11R/JAM-A may result in pathological consequences and disorders in normal cell function. A growing body of evidence points to its role in carcinogenesis and invasiveness, but its tissue-specific pro- or anti-tumorigenic role remains a debated issue. The following review focuses on the F11R/JAM-A tissue-dependent manner in tumorigenesis and metastasis and also discusses the correlation between poor patient clinical outcomes and its aberrant expression. In the future, it will be required to clarify the signaling pathways that are activated or suppressed via the F11R/JAM-A protein in various cancer types to understand its multiple roles in cancer progression and further use it as a novel direct target for cancer treatment.

Sex-dependent Lupus Ruminococcus blautia gnavus strain induction of zonulin-mediated intestinal permeability and autoimmunity

Imbalances in the gut microbiome are suspected as contributors to the pathogenesis of Systemic Lupus Erythematosus, and our studies and others have documented that patients with active Lupus nephritis have expansions of the obligate anaerobe, Ruminococcus blautia gnavus (RG). To investigate whether the RG strains in Lupus patients have in vivo pathogenic properties, we colonized C57BL/6 mice with individual RG strains from healthy adults or those from Lupus patients. These strains had a similar capacity for murine intestinal colonization, in antibiotic-preconditioned specific-pathogen-free, as well as germ-free adults, and their neonatally colonized litters. Lupus-derived RG strains induced high levels of intestinal permeability that was significantly greater in female than male mice, whereas the RG species-type strain (ATCC29149/VPI C7-1) from a healthy donor had little or no effects. Lupus RG strain-induced functional alterations were associated dysregulated occluden transcript production in the ileal wall as well as raised serum levels of zonulin, a regulator of tight junction formation between cells that form the gut barrier. Notably, the level of Lupus RG-induced intestinal permeability was significantly correlated with serum IgG anti RG cell-wall lipoglycan antibodies, and to anti-native DNA autoantibodies that are a biomarker for SLE. Strikingly, gut permeability was completely reversed by oral treatment with larazotide acetate, an octapeptide that is a specific molecular antagonist of zonulin. Taken together, these studies document a molecular pathway by which RG strains from Lupus patients induce a leaky gut and autoimmunity that have been implicated in the pathogenesis of flares of clinical Lupus disease.

Dysregulation of Blood-Brain Barrier and Exacerbated Inflammatory Response in Cx47-Deficient Mice after Induction of EAE

Induction of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), in connexin 32 (Cx32) or Cx47 knockout (KO) mice with deficiency in oligodendrocyte gap junctions (GJs) results in a more severe disease course. In particular, Cx47 KO EAE mice experience an earlier EAE onset and more pronounced disease severity, accompanied by dysregulated pro-inflammatory responses preceding the disease manifestations. In this study, analysis of relevant pro-inflammatory cytokines in wild type EAE, Cx32 KO EAE, and Cx47 KO EAE mice revealed altered expression of Vcam-1 preceding EAE [7 days post injection (dpi)], of Ccl2 at the onset of EAE (12 dpi), and of Gm-csf at the peak of EAE (24 dpi) in Cx47 KO EAE mice. Moreover, Cx47 KO EAE mice exhibited more severe blood-spinal cord barrier (BSCB) disruption, enhanced astrogliosis with defects in tight junction formation at the glia limitans, and increased T-cell infiltration prior to disease onset. Thus, Cx47 deficiency appears to cause dysregulation of the inflammatory profile and BSCB integrity, promoting early astrocyte responses in Cx47 KO EAE mice that lead to a more severe EAE outcome. Further investigation into the role of oligodendrocytic Cx47 in EAE and multiple sclerosis pathology is warranted.

Pals1 prevents Rac1-dependent colorectal cancer cell metastasis by inhibiting Arf6

Loss of apical-basal polarity and downregulation of cell-cell contacts is a critical step during the pathogenesis of cancer. Both processes are regulated by the scaffolding protein Pals1, however, it is unclear whether the expression of Pals1 is affected in cancer cells and whether Pals1 is implicated in the pathogenesis of the disease.Using mRNA expression data and immunostainings of cancer specimen, we show that Pals1 is frequently downregulated in colorectal cancer, correlating with poorer survival of patients. We further found that Pals1 prevents cancer cell metastasis by controlling Rac1-dependent cell migration through inhibition of Arf6, which is independent of the canonical binding partners of Pals1. Loss of Pals1 in colorectal cancer cells results in increased Arf6 and Rac1 activity, enhanced cell migration and invasion in vitro and increased metastasis of transplanted tumor cells in mice. Thus, our data reveal a new function of Pals1 as a key inhibitor of cell migration and metastasis of colorectal cancer cells. Notably, this new function is independent of the known role of Pals1 in tight junction formation and apical-basal polarity.

P059 GB004 drives protective effects on immune cells and epithelial cells using human ex vivo monolayer and co-culture systems

Background GB004 is a small molecule that stabilizes hypoxia inducible factor (HIF-1α), a key transcription factor involved in the cellular responses at the intersection of hypoxia and inflammation. GB004 induction of HIF-1a target genes improves barrier integrity and reduces barrier permeability in rodent models of colitis. Here we investigated gene expression and secretion of soluble factors elicited by GB004 treatment on immune cells and epithelial cells, human monolayer assays and immune co-culture studies to further explore its protective mechanism. Methods In the BioMAP human co-culture assay, cells were treated with GB004 one hour prior to stimulation and incubated for 24–168 hours. Biomarkers, cell viability and proliferation were assessed. Repligut human stem-cell-derived monolayer platforms were used to assess GB004 effects under unstimulated or TNFa stimulation conditions. Barrier integrity was assessed by Transepithelial Endothelial Electric Resistance (TEER), HIF-1a target genes were assessed in cell lysates, and tight junction formation and epithelial monolayer viability were investigated by immunofluorescence staining. Results GB004 demonstrated activity in 11 of 12 systems in the BioMAP human co-culture assay, which covers a range of disease-relevant immune and non-immune mechanisms under stimulation conditions. Collectively, GB004 treatment led to changes in biomarkers associated with inflammation (sTNFa, IL-8, MCP-1, CXCL11), immunomodulation (sIL-17F, sIL-17A), and tissue remodeling (collagen I, collagen IV). GB004 treatment also reduced the proliferative activity in the in vitro lymphocyte assay systems. In the human Repligut intestinal epithelium assay, GB004 significantly reduced cell death and improved barrier integrity in response to pro-inflammatory cytokines. The protective role of GB004 on barrier integrity was further supported by immunostaining experiments demonstrating that GB004 treatment protected epithelial cells from TNFa-induced cell apoptosis, improving both epithelial cell number and tight junction protein ZO-1 staining. Conclusion GB004 modulates key anti-inflammatory and immunomodulatory activities in human co-culture systems. Furthermore, GB004 demonstrates protective effects on human derived monolayer cultures by reducing cell death, promoting tight junction formation, and improving barrier integrity. Targeting both barrier function and local colonic inflammation represents a multi-faceted approach to treatment of inflammatory bowel disease. A phase 2 clinical study of GB004 is ongoing in patients with ulcerative colitis (NCT04556383). Sponsored by GB004, Inc., a wholly owned subsidiary of Gossamer Bio, Inc.

Development of a hollow fibre-based renal module for active transport studies

Understanding the active transport of substrates by the kidney in the renal proximal convoluted tubule is crucial for drug development and for studying kidney diseases. Currently, cell-based assays are applied for this this purpose, however, differences between assays and the body are common, indicating the importance of in vitro–in vivo discrepancies. Several studies have suggested that 3D cell cultures expose cells to a more physiological environments, thus, providing more accurate cell function results. To mimic the renal proximal tubule, we have developed a custom-made renal module (RM), containing a single polypropylene hollow fibre (Plasmaphan P1LX, 3M) that serves as a porous scaffold and compared to conventional Transwell cell-based bidirectional transport studies. In addition, a constant flow of media, exposed cells to a physiological shear stress of 0.2 dyne/cm2. MDCK-Mdr1a cells, overexpressing the rat Mdr1a (P-gp) transporter, were seeded onto the HF membrane surface coated with the basement membrane matrix Geltrex which facilitated cell adhesion and tight junction formation. Cells were then seeded into the HF lumen where attachment and tight junction formation were evaluated by fluorescence microscopy while epithelial barrier integrity under shear stress was shown to be achieved by day 7. qPCR results have shown significant changes in gene expression compared to cells grown on Transwells. Kidney injury marker such as KIM-1 and the hypoxia marker CA9 have been downregulated, while the CD133 (Prominin-1) microvilli marker has shown a fivefold upregulation. Furthermore, the renal transporter P-gp expression has been downregulated by 50%. Finally, bidirectional assays have shown that cells grown in the RM were able to reabsorb albumin with a higher efficiency compared to Transwell cell cultures while efflux of the P-gp-specific substrates Hoechst and Rhodamine 123 was decreased. These results further support the effect of the microenvironment and fluidic shear stress on cell function and gene expression. This can serve as the basis for the development of a microphysiological renal model for drug transport studies.