Renal macrophages monitor and remove particles from urine to prevent tubule obstruction.

When the filtrate of the glomerulus flows through the renal tubular system, various microscopic sediment particles, including mineral crystals, are generated. Dislodging these particles is critical to ensuring the free flow of filtrate, whereas failure to remove them will result in kidney stone formation and obstruction. However, the underlying mechanism for the clearance is unclear. Here, using high-resolution microscopy, we found that the juxtatubular macrophages in the renal medulla constitutively formed transepithelial protrusions and "sampled" urine contents. They efficiently sequestered and phagocytosed intraluminal sediment particles and occasionally transmigrated to the tubule lumen to escort the excretion of urine particles. Mice with decreased renal macrophage numbers were prone to developing various intratubular sediments, including kidney stones. Mechanistically, the transepithelial behaviors of medulla macrophages required integrin ß1-mediated ligation to the tubular epithelium. These findings indicate that medulla macrophages sample urine content and remove intratubular particles to keep the tubular system unobstructed.

Tubulointerstitial nephritis antigen-like 1 from cancer-associated fibroblasts contribute to the progression of diffuse-type gastric cancers through the interaction with integrin ß1.

BACKGROUND: Tumor cells of diffuse-type gastric cancer (DGC) are discohesive and infiltrate into the stroma as single cells or small subgroups, so the stroma significantly impacts DGC progression. Cancer-associated fibroblasts (CAFs) are major components of the tumor stroma. Here, we identified CAF-specific secreted molecules and investigated the mechanism underlying CAF-induced DGC progression. METHODS: We conducted transcriptome analysis for paired normal fibroblast (NF)-CAF isolated from DGC patient tissues and proteomics for conditioned media (CM) of fibroblasts. The effects of fibroblasts on cancer cells were examined by transwell migration and soft agar assays, western blotting, and in vivo. We confirmed the effect of blocking tubulointerstitial nephritis antigen-like 1 (TINAGL1) in CAFs using siRNA or shRNA. We evaluated the expression of TINAGL1 protein in frozen tissues of DGC and paired normal stomach and mRNA in formalin-fixed, paraffin-embedded (FFPE) tissue using RNA in-situ hybridization (RNA-ISH). RESULTS: CAFs more highly expressed TINAGL1 than NFs. The co-culture of CAFs increased migration and tumorigenesis of DGC. Moreover, CAFs enhanced the phosphorylation of focal adhesion kinase (FAK) and mesenchymal marker expression in DGC cells. In an animal study, DGC tumors co-injected with CAFs showed aggressive phenotypes, including lymph node metastasis. However, increased phosphorylation of FAK and migration were reduced by blocking TINAGL1 in CAFs. In the tissues of DGC patients, TINAGL1 was higher in cancer than paired normal tissues and detected with collagen type I alpha 1 chain (COL1A1) in the same spot. Furthermore, high TINAGL1 expression was significantly correlated with poor prognosis in several public databases and our patient cohort diagnosed with DGC. CONCLUSIONS: These results indicate that TINAGL1 secreted by CAFs induces phosphorylation of FAK in DGC cells and promotes tumor progression. Thus, targeting TINAGL1 in CAFs can be a novel therapeutic strategy for DGC.

Integrin beta1 mediates the effect of telocytes on mesenchymal stem cell proliferation and migration in the treatment of acute lung injury.

Co-transplantation of mesenchymal stem cells (MSCs) with telocytes (TCs) was found to have therapeutic effects, although the mechanism of intercellular communication is still unknown. Our current studies aim at exploring the potential molecular mechanisms of TCs interaction and communication with MSCs with a focus on integrin beta1 (ITGB1) in TCs. We found that the co-culture of MSCs with ITGB1-deleted TCs (TCITGB1-ko ) changed the proliferation, differentiation and growth dynamics ability of MSC in responses to LPS or PI3K inhibitor. Changes of MSC proliferation and apoptosis were accompanied with the dysregulation of cytokine mRNA expression in MSCs co-cultured with TCITGB1-ko during the exposure of PI3Kα/δ/ß inhibitor, of which IL-1ß, IL-6 and TNF-α increased, while IFN-γ, IL-4 and IL-10 decreased. The responses of PI3K p85, PI3K p110 and pAKT of MSCs co-cultured with TCITGB1-ko to LPS or PI3K inhibitor were opposite to those with ITGB1-presented TCs. The intraperitoneal injection of TCITGB1-ko , TCvector or MSCs alone, as well as the combination of MSCs with TCITGB1-ko or TCvector exhibited therapeutic effects on LPS-induced acute lung injury. Thus, our data indicate that telocyte ITGB1 contributes to the interaction and intercellular communication between MSCs and TCs, responsible for influencing other cell phenomes and functions.

LAPTM4B-35 promotes cancer cell migration via stimulating integrin beta1 recycling and focal adhesion dynamics.

Metastasis is the main cause of cancer patients' death despite tremendous efforts invested in developing the related molecular mechanisms. During cancer cell migration, cells undergo dynamic regulation of filopodia, focal adhesion, and endosome trafficking. Cdc42 is imperative for maintaining cell morphology and filopodia, regulating cell movement. Integrin beta1 activates on the endosome, the majority of which distributes itself on the plasma membrane, indicating that endocytic trafficking is essential for this activity. In cancers, high expression of lysosome-associated protein transmembrane 4B (LAPTM4B) is associated with poor prognosis. LAPTM4B-35 has been reported as displaying plasma membrane distribution and being associated with cancer cell migration. However, the detailed mechanism of its isoform-specific distribution and whether it relates to cell migration remain unknown. Here, we first report and quantify the filopodia localization of LAPTM4B-35: mechanically, that specific interaction with Cdc42 promoted its localization to the filopodia. Furthermore, our data show that LAPTM4B-35 stabilized filopodia and regulated integrin beta1 recycling via interaction and cotrafficking on the endosome. In our zebrafish xenograft model, LAPTM4B-35 stimulated the formation and dynamics of focal adhesion, further promoting cancer cell dissemination, whereas in skin cancer patients, LAPTM4B level correlated with poor prognosis. In short, this study establishes an insight into the mechanism of LAPTM4B-35 filopodia distribution, as well as into its biological effects and its clinical significance, providing a novel target for cancer therapeutics development.

Fertilization, but Not Post-Implantation Development, Can Occur in the Absence of Sperm and Oocyte Beta1 Integrin in Mice.

Fertilization is a complex process that requires successive stages and culminates in the adhesion/fusion of gamete membranes. If the question of the involvement of oocyte integrins has been swept away by deletion experiments, that of the involvement of sperm integrins remains to be further characterized. In the present study, we addressed the question of the feasibility of sperm-oocyte adhesion/fusion and early implantation in the absence of sperm ß1 integrin. Males and females with ß1 integrin-depleted sperm and oocytes were mated, and fertilization outcome was monitored by a gestational ultrasound analysis. Results suggest that although the sperm ß1 integrin participates in gamete adhesion/fusion, it is dispensable for fertilization in mice. However, sperm- and/or oocyte-originated integrin ß1 is essential for post-implantation development. Redundancy phenomena could be at the origin of a compensatory expression or alternative dimerization pattern.

TBC1 domain family member 23 interacts with Ras-related protein Rab-11A to promote poor prognosis of non-small-cell lung cancer via ß1-integrin.

Non-small-cell lung cancer (NSCLC) accounts for approximately 80% of lung cancer cases. TBC1D23, a member of the TBC/RABGAP family, is widely expressed in human tissues; however, its role in NSCLC is currently unknown. Immunohistochemical analysis was conducted on 173 paraffin-embedded lung tissue sections from patients with NSCLC from 2014 to 2018 at the First Affiliated Hospital of China Medical University. MTT, colony formation assay, cell cycle assay, scratch assay, transwell assay, Western blotting and real-time PCR were employed on multiple NSCLC cell lines modified to knock down or overexpress TBC1D23/RAB11A. Immunoprecipitation, immunoprecipitation-mass spectrometry, immunofluorescence and flow cytometry were performed to explore the interaction between TBC1D23 and RAB11A and TBC1D23 involvement in the interaction between RAB11A and ß1 integrin in the para-nucleus. TBC1D23 was correlated with tumour size, differentiation degree, metastasis, TNM stage and poor prognosis. TBC1D23 was involved in the interaction between RAB11A and ß1 integrin in the para-nucleus, thus activating the ß1 integrin/FAK/ERK signalling pathway to promote NSCLC. Furthermore, TBC1D23 promoted NSCLC progression by inducing cell proliferation, migration and invasion. This study indicated the relationship between TBC1D23 expression and the adverse clinicopathological characteristics of patients with NSCLC, suggesting that TBC1D23 may be an important target for NSCLC treatment.

Collagen receptor cross-talk determines α-smooth muscle actin-dependent collagen gene expression in angiotensin II-stimulated cardiac fibroblasts.

Excessive collagen deposition by myofibroblasts during adverse cardiac remodeling leads to myocardial fibrosis that can compromise cardiac function. Unraveling the mechanisms underlying collagen gene expression in cardiac myofibroblasts is therefore an important clinical goal. The collagen receptors, discoidin domain receptor 2 (DDR2), a collagen-specific receptor tyrosine kinase, and integrin-ß1, are reported to mediate tissue fibrosis. Here, we probed the role of DDR2-integrin-ß1 cross-talk in the regulation of collagen α1(I) gene expression in angiotensin II (Ang II)-stimulated cardiac fibroblasts. Results from gene silencing/overexpression approaches, electrophoretic mobility shift assays, and ChIP revealed that DDR2 acts via extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2 MAPK)-dependent transforming growth factor-ß1 (TGF-ß1) signaling to activate activator protein-1 (AP-1) that in turn transcriptionally enhances the expression of collagen-binding integrin-ß1 in Ang II-stimulated cardiac fibroblasts. The DDR2-integrin-ß1 link was also evident in spontaneously hypertensive rats and DDR2-knockout mice. Further, DDR2 acted via integrin-ß1 to regulate α-smooth muscle actin (α-SMA) and collagen type I expression in Ang II-exposed cardiac fibroblasts. Downstream of the DDR2-integrin-ß1 axis, α-SMA was found to regulate collagen α1(I) gene expression via the Ca2+ channel, transient receptor potential cation channel subfamily C member 6 (TRPC6), and the profibrotic transcription factor, Yes-associated protein (YAP). This finding indicated that fibroblast-to-myofibroblast conversion is mechanistically coupled to collagen expression. The observation that collagen receptor cross-talk underlies α-SMA-dependent collagen type I expression in cardiac fibroblasts expands our understanding of the complex mechanisms involved in collagen gene expression in the heart and may be relevant to cardiac fibrogenesis.

Smilax glabra Roxb. Inhibits Collagen Induced Adhesion and Migration of PC3 and LNCaP Prostate Cancer Cells through the Inhibition of Beta 1 Integrin Expression.

Smilax glabra Roxb. (SGR) has been used as a traditional medicine for brucellosis and syphilis. In this study, we investigated whether nontoxicological levels of water extract of SGR (WESGR) are effective for suppressing steps in the progression of prostate cancer, such as collagen-mediated migration and adhesion and identified the target molecule responsible for such effects. We found that nontoxicological levels of WESGR did not attenuate PC3 and LNCaP cell adhesion to serum but did significantly do so with collagen. In addition, using the Boyden chamber assay, we found that nontoxicological levels of WESGR did not inhibit the migration of PC3 and LNCaP cells to a serum-coated area but did significantly attenuate migration to a collagen-coated area. Interestingly, the expression of α2ß1 integrin, a known receptor of collagen, was not affected by ectopic administration of WESGR. However, WESGR significantly attenuated the expression of ß1 integrin, but not α2 integrin when PC3 and LNCaP cells were placed on a collagen-coated plate, resulting in attenuation of focal adherent kinase phosphorylation. Finally, 5-O-caffeoylquinic acid was determined as a functional single component which is responsible for antiprostate cancer effects of WESGR. Taken together, our results suggest a novel molecular mechanism for WESGR-mediated antiprostate cancer effects at particular steps such as with migration and adhesion to collagen, and it could provide the possibility of therapeutic use of WESGR against prostate cancer progression.

Differential Expression of Integrin ß1 in Two Brain Injury Models of Rats.

ABSTRACT: Objective To study the characteristics of positive expression of integrin ß1 in the rat brain tissue of two kinds of traumatic brain injury models and to explore the feasibility of inferring the mode of traumatic brain injury using the positive expression of integrin ß1. Methods The occipital region of rats was hit by hydraulic impact method and pendulum striking method to produce two closed brain injury models of linear and rotation acceleration respectively, then 120 SD rats were randomly divided into linear acceleration injury group, rotation acceleration injury group, sham operation group and normal control group. Immunohistochemistry staining and Western blotting method were used to detect the positive expression of integrin ß1 in different parts of the brain tissue at 30 min, 3 h, 6 h, 12 h, 3 d and 7 d after rat injury. The data was processed statistically by SPSS 18.0 software. Results The positive expression of integrin ß1 was detected 30 min after brain injury and reached the peak 6 h after brain injury. With the extension of injury time, the expression tended to enhance. At the same time points after injury, the differences in the positive expression of integrin ß1 between the linear acceleration injury group and the rotation acceleration injury group in the occipital strike point and thalamus had no statistical significance （ P>0.05）, but the differences in the expression of integrin ß1 in the frontal lobe and brain stem had statistical significance （P<0.05）. Conclusion The characteristics of positive expression of integrin ß1 in brain tissue can be used to infer the strike point and the manner of injury and has application value for the reconstruction of craniocerebral injury process.

PEGylated TiO2 nanoparticles mediated inhibition of cell migration via integrin beta 1.

Nanoparticles (NPs) elicit various physiological responses in cellular environment, and the effect of NPs on cell migration is of high interest. In this work, the effects of NPs on cell migration and their possible mechanisms were studied. Here, we showed that after exposure to pegylated titanium dioxide nanoparticles (TiO2-PEG NPs, where PEG stands for the polyethylene glycol), NCI-H292 cells exhibited slower migration than control cells. Furthermore, larger NPs inhibited cell migration much stronger than smaller NPs. Following NP exposure, the cells showed decreased expression of integrin beta 1 and phosphorylated focal adhesion kinase (pFAK), and disrupted F-actin structures. We demonstrated that a possible mechanism involved NP-mediated promotion of the lysosomal degradation of integrin beta 1, thus leading to reduced expression of pFAK and cytoskeletal disruption and inhibited cell migration. Therefore, our results showed that inhibition of NCI-H292 cell migration by NPs is mediated through integrin beta 1, which provides useful information for the application of NPs in cancer therapy and related fields.

Phagocytic Receptors Activate Syk and Src Signaling during Borrelia burgdorferi Phagocytosis.

Phagocytosis of the Lyme disease-causing pathogen Borrelia burgdorferi has been shown to be important for generating an inflammatory response to the pathogen. As a result, understanding the mechanisms of phagocytosis has been an area of great interest in the field of Lyme disease. Several cell surface receptors that participate in B. burgdorferi phagocytosis have been reported, including the scavenger receptor MARCO and integrin α3ß1. We sought to define the mechanisms by which these receptors mediate phagocytosis and to identify signaling pathways activated downstream of these receptors upon contact with B. burgdorferi We identified both Syk and Src signaling pathways as ones that participate in B. burgdorferi phagocytosis and the resulting cytokine activation. In our studies, we found that both MARCO and integrin ß1 play a role in the activation of the Src kinase pathway. However, only integrin ß1 participates in the activation of Syk. Interestingly, the integrin activates Syk without the help of the signaling adaptor Dap12 or FcRγ. Thus, we report that multiple pathways participate in B. burgdorferi internalization and that different cell surface receptors act simultaneously in cooperation and independently to mediate phagocytosis.

Fibulin-5 regulates keloid-derived fibroblast-like cells through integrin beta-1.

OBJECTIVE: Keloid scar is pathological tissue that appears after skin injury, and that is more aggressive than hypertrophic scars. Keloid scars are characterized by increased proliferation of fibroblast-like cells (FLCs) and the accumulation of extracellular matrix, mainly collagen. Fibulin-5, a glycoprotein secreted by many cell types, is a component of the extracellular matrix. We investigated the effect of fibulin-5 on the adhesion and proliferation of FLCs derived from keloid scars and the role of integrin beta-1 in these activities. METHODS: Fibroblast-like cells were isolated from six keloid scars and cultured on plates coated with fibulin-5 or with gelatin. Cells were incubated for 72-96 h to examine proliferation rates and incubated for 240 min, with washings at 20, 40, 60, 90, 120, 180 min, to assess adhesion rates. To examine the role of integrin beta-1, the anti-human integrin beta-1 (CD29) antibody was added to the culture medium. RESULTS: Fibroblast-like cells from keloids cultured on a fibulin-5-coated surface showed a significantly reduced proliferation rate and a delayed adhesion rate, compared to cells cultured on gelatin-coated dishes. Adherence of these cells to fibulin-5 pre-coated wells was significantly reduced in the presence of anti-human integrin beta-1 (CD29) antibodies. Our current findings are similar to previously observed reduced proliferation in vascular smooth muscle cells overexpressing fibulin-5. We did not test the effects of fibulin-5 on normal fibroblasts. CONCLUSION: This study demonstrates the pivotal role of the extracellular protein, fibulin-5, on the adhesion and proliferation of human keloid-derived cells, through binding to integrin beta-1.

Developmentally regulated collagen/integrin interactions confer adhesive properties to early postnatal neural stem cells.

BACKGROUND: It is becoming increasingly apparent that the extracellular matrix acts as an important regulator of the neural stem niche. Previously we found that neural stem and progenitor cells (NSPCs) derived from the early postnatal subventricular zone of mice adhere to a collagen/hyaluronan hydrogel, whereas NSPCs from the adult and embryonic brain do not. METHODS: To examine the specific adhesive properties of young stem cells in more detail, NSPCs isolated from embryonic, postnatal day 6 (P6), and adult mouse brains were cultured on collagen I. RESULTS: Early postnatal NSPCs formed paxillin-positive focal adhesions on collagen I, and these adhesions could be prevented by an antibody that blocked integrin ß1. Furthermore, we found the corresponding integrin alpha subunits α2 and α11 levels to be highest at the postnatal stage. Gene ontology analysis of differentially expressed genes showed higher expression of transcripts involved in vasculature development and morphogenesis in P6 stem cells, compared to adult. CONCLUSIONS: The ability to interact with the extracellular matrix differs between postnatal and adult NSPCs. GENERAL SIGNIFICANCE: Our observations that the specific adhesive properties of early postnatal NSPCs, which are lost in the adult brain, can be ascribed to the integrin subunits expressed by the former furthering our understanding of the developing neurogenic niche. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.

The integrin beta1 modulator Tirofiban prevents adipogenesis and obesity by the overexpression of integrin-linked kinase: a pre-clinical approach in vitro and in vivo.

BACKGROUND: Obesity is caused by the enlargement of the white adipose tissue (WAT) depots, characterized by the hypertrophic enlargement of malfunctioning adipocytes within WAT which increases the storage of triglycerides (TG) in the lipid droplets (LD). Adipogenesis pathways as well as the expression and activity of some extracellular matrix receptors integrins are upregulated. Integrinß1 (INTB1) is the main isoform involved in WAT remodeling during obesity and insulin resistance-related diseases. We recently described Integrin Linked Kinase (ILK), a scaffold protein recruited by INTB1, as an important mediator of WAT remodeling and insulin resistance. As the few approved drugs to fight obesity have brought long-term cardiovascular side effects and given that the consideration of INTB1 and/or ILK modulation as anti-obesogenic strategies remains unexplored, we aimed to evaluate the anti-obesogenic capacity of the clinically approved anticoagulant Tirofiban (TF), stated in preclinical studies as a cardiovascular protector. METHODS: Fully differentiated adipocytes originating from C3H10T1/2 were exposed to TF and were co-treated with specific INTB1 blockers or with siRNA-based knockdown ILK expression. Lipid-specific dyes were used to determine the TG content in LD. The genetic expression pattern of ILK, pro-inflammatory cytokines (MCP1, IL6), adipogenesis (PPARγ, Leptin), thermogenesis (UCP1), proliferation (PCNA), lipid metabolism (FASN, HSL, ATGL), and metabolite transporters (FABP4, FAT, AQP7) were detected using quantitative PCR. Cytoskeletal actin polymerization was detected by confocal microscopy. Immunoblotting was performed to detect INTB1 phosphorylation at Thr788/9 and ILK activity as phosphorylation levels of protein kinase B (AKT) in Ser473 and glycogen synthase kinase 3ß (GSK3ß) at Ser9. TF was intraperitoneally administered once per day to wildtype and ILK knockdown mice (cKDILK) challenged with a high-fat diet (HFD) or control diet (STD) for 2 weeks. Body and WAT weight gains were compared. The expression of ILK and other markers was determined in the visceral epididymal (epi) and inguinal subcutaneous (sc) WAT. RESULTS: TF reduced TG content and the expression of adipogenesis markers and transporters in adipocytes, while UCP-1 expression was increased and the expression of lipases, cytokines or PCNA was not affected. Mechanistically, TF rapidly increased and faded the intracellular phosphorylation of INTB1 but not AKT or GSK3ß. F-actin levels were rapidly decreased, and INTB1 blockade avoided the TF effect. After 24 h, ILK expression and phosphorylation rates of AKT and GSK3ß were upregulated, while ILK silencing increased TG content. INTB1 blockade and ILK silencing avoided TF effects on the TG content and the transcriptional expression of PPARγ and UCP1. In HFD-challenged mice, the systemic administration of TF for several days reduced the weight gain on WAT depots. TF reduced adipogenesis and pro-inflammatory biomarkers and increased lipolysis markers HSL and FAT in epiWAT from HFD, while increased UCP1 in scWAT. In both WATs, TF upregulated ILK expression and activity, while no changes were observed in other tissues. In HFD-fed cKDILK, the blunted ILK in epiWAT worsened weight gain and avoided the anti-obesogenic effect of in vivo TF administration. CONCLUSIONS: ILK downregulation in WAT can be considered a biomarker of obesity establishment. Via an INTB1-ILK axis, TF restores malfunctioning hypertrophied WAT by changing the expression of adipocyte-related genes, increasing ILK expression and activity, and reducing TG storage. TF prevents obesity, a property to be added to its anticoagulant and cardiovascular protective advantages.

Investigation of the Presence of Integrin Alpha-3 and Beta-1 Receptors on Tumor Tissue, Metastatic Lymph Node and Normal Tissue in Thyroid Cancer.

Objectives: The important roles of integrins in tumor invasion, migration and proliferation are well known. In this study, we investigated the presence of integrin α3 and ß1 receptors in tumor tissue, metastatic lymph node (LN) and normal thyroid tissue of patients diagnosed with thyroid cancer (TCa) and showed the prognostic and diagnostic value of these molecules as well as peptide-receptor. Methods: Sixty-one patients with TCa were included in this study. The presence of integrin α3 and ß1 expression was investigated by immunohistochemical methods from tumor tissue after total thyroidectomy. TNM system was used in tumor staging. The relationship between prognostic properties such as tumor size, LN metastasis, capsular invasion and the presence of integrin α3 and ß1 expression was investigated. Results: Classical type papillary TCa was the most common subtype in our study group with 31.1%. Integrin ß1 was expressed in 4.9% (n=3) of normal tissue, 57.4% (n=35) of tumor tissue and 16.4% (n=10) of metastatic LN; integrin α3 was expressed in 50.8% (n=31) of normal tissue, 67.2% (n=41) of tumor tissue and 9.8% (n=6) metastatic LN. Integrin ß1 expression was observed 21.3% (n=13), integrin α3 in 14.8% (n=9) and integrin α3 and ß1 expression in 36.1% (n=22). Integrin ß1 expression increased statistically significantly in the presence of LN metastasis and capsular invasion (p=0.022, 0.014, respectively). Furthermore, the expression of integrin α3 was found to be statistically significant in primary tumors of patients with LN metastasis (p=0.045). Conclusion: Our study showed a significant increase in integrin α3 and ß1 expression in LN metastasis or thyroid capsule invasion in tumor. Thus, it appears that the demonstration of the presence of integrin α3 and ß1 expression in TCa is not only a prognostic biomarker but also has value as a potential theranostic target with peptide-bound radioactive agents.

Knockdown of CCL28 inhibits endometriosis stromal cell proliferation and invasion via ERK signaling pathway inactivation.

Endometriosis (EM), the presence of functional endometrial glands and stroma outside the uterine cavity, is a common gynecological disorder. At present, the pathogenesis of EM has not been fully elucidated, so there is still a lack of effective therapy. The present study aimed to explore the role of C­C motif chemokine ligand 28 (CCL28) and its underlying mechanism in endometrial stromal cells to propose a novel therapy for EM treatment. The expression of CCL28 and CC chemokine receptor 10 (CCR10) were examined. After CCL28 knockdown or overexpression by lentivirus infection, cell proliferation and invasion were measured. It was revealed that compared with normal, the expression levels of CCL28 and CCR10 were significantly elevated in endometrial tissues of patients with EM. Knockdown of CCL28 in endometrial stromal cells significantly suppressed cell proliferation and invasion, and this was accompanied by significantly reduced expression levels of CCR10, MMP2, MMP9, integrin ß1 (ITGB1) and phosphorylated (p)­ERK/ERK ratio. The addition of the CCL28 recombinant protein had an opposite effect to CCL28 downregulation. Furthermore, the ERK inhibitor, PD98059, reduced CCL28­induced cell proliferation and invasion, as well as the expression levels of MMP2, MMP9, ITGB1 and p­ERK. Therefore, the present study indicated that CCL28 may contribute to the progression of EM by regulating MMP2, MMP9 and ITGB1 expression and function via the activation of the ERK signaling pathway.

Genome-wide CRISPR screen identified a role for commander complex mediated ITGB1 recycling in basal insulin secretion.

OBJECTIVES: Pancreatic beta cells secrete insulin postprandially and during fasting to maintain glucose homeostasis. Although glucose-stimulated insulin secretion (GSIS) has been extensively studied, much less is known about basal insulin secretion. Here, we performed a genome-wide CRISPR/Cas9 knockout screen to identify novel regulators of insulin secretion. METHODS: To identify genes that cell autonomously regulate insulin secretion, we engineered a Cas9-expressing MIN6 subclone that permits irreversible fluorescence labeling of exocytic insulin granules. Using a fluorescence-activated cell sorting assay of exocytosis in low glucose and high glucose conditions in individual cells, we performed a genome-wide CRISPR/Cas9 knockout screen. RESULTS: We identified several members of the COMMD family, a conserved family of proteins with central roles in intracellular membrane trafficking, as positive regulators of basal insulin secretion, but not GSIS. Mechanistically, we show that the Commander complex promotes insulin granules docking in basal state. This is mediated, at least in part, by its function in ITGB1 recycling. Defective ITGB1 recycling reduces its membrane distribution, the number of focal adhesions and cortical ELKS-containing complexes. CONCLUSIONS: We demonstrated a previously unknown function of the Commander complex in basal insulin secretion. We showed that by ITGB1 recycling, Commander complex increases cortical adhesions, which enhances the assembly of the ELKS-containing complexes. The resulting increase in the number of insulin granules near the plasma membrane strengthens basal insulin secretion.

TMEM182 interacts with integrin beta 1 and regulates myoblast differentiation and muscle regeneration.

BACKGROUND: Transmembrane proteins are vital for intercellular signalling and play important roles in the control of cell fate. However, their physiological functions and mechanisms of action in myogenesis and muscle disorders remain largely unexplored. It has been found that transmembrane protein 182 (TMEM182) is dramatically up-regulated during myogenesis, but its detailed functions remain unclear. This study aimed to analyse the function of TMEM182 during myogenesis and muscle regeneration. METHODS: RNA sequencing, quantitative real-time polymerase chain reaction, and immunofluorescence approaches were used to analyse TMEM182 expression during myoblast differentiation. A dual-luciferase reporter assay was used to identify the promoter region of the TMEM182 gene, and a chromatin immunoprecipitation assay was used to investigate the regulation TMEM182 transcription by MyoD. We used chickens and TMEM182-knockout mice as in vivo models to examine the function of TMEM182 in muscle growth and muscle regeneration. Chickens and mouse primary myoblasts were used to extend the findings to in vitro effects on myoblast differentiation and fusion. Co-immunoprecipitation and mass spectrometry were used to identify the interaction between TMEM182 and integrin beta 1 (ITGB1). The molecular mechanism by which TMEM182 regulates myogenesis and muscle regeneration was examined by Transwell migration, cell wound healing, adhesion, glutathione-S-transferse pull down, protein purification, and RNA immunoprecipitation assays. RESULTS: TMEM182 was specifically expressed in skeletal muscle and adipose tissue and was regulated at the transcriptional level by the myogenic regulatory factor MyoD1. Functionally, TMEM182 inhibited myoblast differentiation and fusion. The in vivo studies indicated that TMEM182 induced muscle fibre atrophy and delayed muscle regeneration. TMEM182 knockout in mice led to significant increases in body weight, muscle mass, muscle fibre number, and muscle fibre diameter. Skeletal muscle regeneration was accelerated in TMEM182-knockout mice. Furthermore, we revealed that the inhibitory roles of TMEM182 in skeletal muscle depend on ITGB1, an essential membrane receptor involved in cell adhesion and muscle formation. TMEM182 directly interacted with ITGB1, and this interaction required an extracellular hybrid domain of ITGB1 (aa 387-470) and a conserved region (aa 52-62) within the large extracellular loop of TMEM182. Mechanistically, TMEM182 modulated ITGB1 activation by coordinating the association between ITGB1 and laminin and regulating the intracellular signalling of ITGB1. Myogenic deletion of TMEM182 increased the binding activity of ITGB1 to laminin and induced the activation of the FAK-ERK and FAK-Akt signalling axes during myogenesis. CONCLUSIONS: Our data reveal that TMEM182 is a novel negative regulator of myogenic differentiation and muscle regeneration.

Gremlin 2 suppresses differentiation of stem/progenitor cells in the human skin.

INTRODUCTION: The skin is comprised of various kinds of cells and has three layers, the epidermis, dermis and subcutaneous adipose tissue. Stem cells in each tissue duplicate themselves and differentiate to supply new cells that function in the tissue, and thereby maintain the tissue homeostasis. In contrast, senescent cells accumulate with age and secrete senescence-associated secretory phenotype (SASP) factors that impair surrounding cells and tissues, which lowers the capacity to maintain homeostasis in each tissue. Previously, we found Gremlin 2 (GREM2) as a novel SASP factor in the skin and reported that GREM2 suppressed the differentiation of adipose-derived stromal/stem cells. In the present study, we investigated the effects of GREM2 on stem cells in the epidermis and dermis. METHODS: To examine whether GREM2 expression and the differentiation levels in the epidermis and dermis are correlated, the expressions of GREM2, stem cell markers, an epidermal differentiation marker Keratin 10 (KRT10) and a dermal differentiation marker type 3 procollagen were examined in the skin samples (n = 14) randomly chosen from the elderly where GREM2 expression level is high and the individual differences of its expression are prominent. Next, to test whether GREM2 affects the differentiation of skin stem cells, cells from two established lines (an epidermal and a dermal stem/progenitor cell model) were cultured and induced to differentiate, and recombinant GREM2 protein was added. RESULTS: In the human skin, the expression levels of GREM2 varied among individuals both in the epidermis and dermis. The expression level of GREM2 was not correlated with the number of stem cells, but negatively correlated with those of both an epidermal and a dermal differentiation markers. The expression levels of epidermal differentiation markers were significantly suppressed by the addition of GREM2 in the three-dimensional (3D) epidermis generated with an epidermal stem/progenitor cell model. In addition, by differentiation induction, the expressions of dermal differentiation markers were induced in cells from a dermal stem/progenitor cell model, and the addition of GREM2 significantly suppressed the expressions of the dermal differentiation markers. CONCLUSIONS: GREM2 expression level did not affect the numbers of stem cells in the epidermis and dermis but affects the differentiation and maturation levels of the tissues, and GREM2 suppressed the differentiation of stem/progenitor cells in vitro. These findings suggest that GREM2 may contribute to the age-related reduction in the capacity to maintain skin homeostasis by suppressing the differentiation of epidermal and dermal stem/progenitor cells.

Platelet endothelial aggregation receptor-1 regulates bovine muscle satellite cell migration and differentiation via integrin beta-1 and focal adhesion kinase.

PEAR1 is highly expressed at bovine MDSC differentiation. However, its biological function remains unclear. Western blotting results showed that PEAR1 increased between day 0 and day 2 of cell differentiation and decreased from day 3. Moreover, scratch test showed that wound healing rate increased after PEAR1 overexpression and decreased upon its suppression. Meanwhile, we found that, upon PEAR1 induction, both the expression of the focal adhesion-associated and MyoG, and the myotube fusion rate increased. However, when PEAR1 was suppressed, opposite results were obtained. Immunoprecipitation revealed an association between PEAR1 and ITGB1. Notably, inhibition of FAK and ITGB1 repressed cell differentiation. In conclusion, our study indicated that PEAR1 is involved in the regulation of bovine MDSC migration and differentiation.