Podocalyxin promotes the formation of compact and chemoresistant cancer spheroids in high grade serous carcinoma.

High grade serous carcinoma (HGSC) metastasises primarily intraperitoneally via cancer spheroids. Podocalyxin (PODXL), an anti-adhesive transmembrane protein, has been reported to promote cancer survival against chemotherapy, however its role in HGSC chemoresistance is unclear. This study investigated whether PODXL plays a role in promoting chemoresistance of HGSC spheroids. We first showed that PODXL was expressed variably in HGSC patient tissues (n = 17) as well as in ovarian cancer cell lines (n = 28) that are more likely categorised as HGSC. We next demonstrated that PODXL-knockout (KO) cells proliferated more slowly, formed less compact spheroids and were more fragile than control cells. Furthermore, when treated with carboplatin and examined for post-treatment recovery, PODXL-KO spheroids showed significantly poorer cell viability, lower number of live cells, and less Ki-67 staining than controls. A similar trend was also observed in ascites-derived primary HGSC cells (n = 6)-spheroids expressing lower PODXL formed looser spheroids, were more vulnerable to fragmentation and more sensitive to carboplatin than spheroids with higher PODXL. Our studies thus suggests that PODXL plays an important role in promoting the formation of compact/hardy HGSC spheroids which are more resilient to chemotherapy drugs; these characteristics may contribute to the chemoresistant nature of HGSC.

Simple Binding and Dissociation of a Sialoglycoprotein Using Boronic Acid-Modified Functional Interfaces on Microparticles.

An overexpression of sialic acid is an indicator of metastatic cancer, and selective detection of sialic acid shows potential for cancer diagnosis. Boronic acid is a promising candidate for this purpose because of its ability to specifically bind to sialic acid under acidic conditions. Notably, the binding strength can be easily modulated by adjusting the pH, which allows for a simple dissociation of the bound sialic acid. In this study, we developed 5-boronopicolinic acid (5-BPA)-modified magnetic particles (BMPs) to selectively capture sialic acid biomolecules. We successfully captured fetuin, a well-known sialoglycoprotein, on BMPs at >104 molecules/particle using an acetate buffer (pH 5.0). Facile dissociation then occurred when the system was changed to a pH 7.6 phosphate buffer. This capture-and-release process could be repeated at least five times. Moreover, this system could enrich fetuin by more than 20 times. In summary, BMPs are functional particles for facile purification and concentration through the selective capture of sialic acid proteins and can improve detection sensitivity compared with conventional methods. This technology shows potential for the detection of sialic acid overexpression by biological particles.

Bone sialoprotein promotes lung cancer osteolytic bone metastasis via MMP14-dependent mechanisms.

Bone metastases during lung cancer are common. Bone sialoprotein (BSP), a non-collagenous bone matrix protein, plays important functions in bone mineralization processes and in integrin-mediated cell-matrix interactions. Importantly, BSP induces bone metastasis in lung cancer, but the underlying mechanisms remain unclear. This study therefore sought to determine the intracellular signaling pathways responsible for BSP-induced migration and invasion of lung cancer cells to bone. Analyses of the Kaplan-Meier, TCGA, GEPIA and GENT2 databases revealed that high levels of BSP expression in lung tissue samples were associated with significantly decreased overall survival (hazard ratio = 1.17; p = 0.014) and with a more advanced clinical disease stage (F-value = 2.38, p < 0.05). We also observed that BSP-induced stimulation of matrix metalloproteinase (MMP)-14 promoted lung cancer cell migration and invasion via the PI3K/AKT/AP-1 signaling pathway. Notably, BSP promoted osteoclastogenesis in RAW 264.7 cells exposed to RANKL and BSP neutralizing antibody reduced osteoclast formation in conditioned medium (CM) from lung cancer cell lines. Finally, at 8 weeks after mice were injected with A549 cells or A549 BSP shRNA cells, the findings revealed that the knockdown of BSP expression significantly reduced metastasis to bone. These findings suggest that BSP signaling promotes lung bone metastasis via its direct downstream target gene MMP14, which reveals a novel potential therapeutic target for lung cancer bone metastases.

AAV6-Mediated Gene Therapy Prevents Developmental Dentin Defects in a Dentinogenesis Imperfecta Type â ¢ Mouse Model.

Dentin is a major type of hard tissue of teeth and plays essential roles for normal tooth function. Odontoblasts are responsible for dentin formation. Mutations or deficiency in various genes affect the differentiation of odontoblasts, leading to irreversible dentin developmental defects in animals and humans. Whether such dentin defects can be reversed by gene therapy for odontoblasts remains unknown. In this study, we compare the infection efficiencies of six commonly used adeno-associated virus (AAV) serotypes (AAV1, AAV5, AAV6, AAV8, AAV9, and AAVDJ) in cultured mouse odontoblast-like cells (OLCs). We show that AAV6 serotype infects OLCs with the highest efficiency among the six AAVs. Two cellular receptors, which are able to recognize AAV6, AAV receptor (AAVR), and epidermal growth factor receptor (EGFR), are strongly expressed in the odontoblast layer of mouse teeth. After local administration to mouse molars, AAV6 infects the odontoblast layer with high efficiency. Furthermore, AAV6-Mdm2 was successfully delivered to teeth and prevents the defects in odontoblast differentiation and dentin formation in Mdm2 conditional knockout mice (a mouse model of dentinogenesis imperfecta type Ⅲ). These results suggest that AAV6 can serve as a reliable and efficient vehicle for gene delivery to odontoblasts through local injection. In addition, human OLCs were also successfully infected by AAV6 with high efficiency, and both AAVR and EGFR are strongly expressed in the odontoblast layer of extracted human developing teeth. These findings suggest that AAV6-mediated gene therapy through local injection may be a promising treatment approach for hereditary dentin disorders in humans.

Spatial regulation of the glycocalyx component podocalyxin is a switch for prometastatic function.

The glycocalyx component and sialomucin podocalyxin (PODXL) is required for normal tissue development by promoting apical membranes to form between cells, triggering lumen formation. Elevated PODXL expression is also associated with metastasis and poor clinical outcome in multiple tumor types. How PODXL presents this duality in effect remains unknown. We identify an unexpected function of PODXL as a decoy receptor for galectin-3 (GAL3), whereby the PODXL-GAL3 interaction releases GAL3 repression of integrin-based invasion. Differential cortical targeting of PODXL, regulated by ubiquitination, is the molecular mechanism controlling alternate fates. Both PODXL high and low surface levels occur in parallel subpopulations within cancer cells. Orthotopic intraprostatic xenograft of PODXL-manipulated cells or those with different surface levels of PODXL define that this axis controls metastasis in vivo. Clinically, interplay between PODXL-GAL3 stratifies prostate cancer patients with poor outcome. Our studies define the molecular mechanisms and context in which PODXL promotes invasion and metastasis.

Biodentine but not MTA induce DSPP expression of dental pulp cells with different severity of LPS-induced inflammation.

OBJECTIVES: To explore the inflammatory and differentiation response in inflamed dental pulp cells (DPCs) induced by lipopolysaccharide (LPS) under different conditions with Biodentine and mineral trioxide aggregate (MTA) treatment. MATERIALS AND METHODS: DPCs were treated with 0.001-1 µg/mL LPS for different periods to induce inflammation. Normal and inflamed DPCs were further treated with 0.14 mg/mL Biodentine or 0.13 mg/mL MTA for different periods. mRNA expression level of IL-6, IL-8 and ALP were analysed by qPCR. DSPP protein expression was detected by western blot. The data were analysed by the Mann-Whitney test, unpaired t test or two-way ANOVA. RESULTS: After treatment for different times and with different concentrations of LPS, different severity of pulp inflammation was revealed by the expressions of IL-6 and IL-8. Higher concentrations of LPS induced higher IL-6 and IL-8 expressions, and these expressions first increased and then decreased (p < 0.0001). At 96 and 192 h, Biodentine significantly suppressed IL-6 expression in both normal and inflamed DPCs (p < 0.05). At 48 and 96 h, Biodentine suppressed ALP expression in both normal and inflamed DPCs (p < 0.05). At 48 and 96 h, Biodentine induced DSPP expressions in both normal and inflamed DPCs (p < 0.05). CONCLUSION: Biodentine enhanced more DSPP differentiation of both normal and inflamed DPCs under different treatment durations than MTA. CLINICAL RELEVANCE: The prognosis of vital pulp therapy may depend on the severity of pulp inflammation which is difficult to be determined in clinical settings. Therefore, Biodentine may enhance odontogenic differentiation in different severity of pulp inflammation imply its clinical indications.

A WWP2-PTEN-KLF5 signaling axis regulates odontoblast differentiation and dentinogenesis in mice.

WW domain-containing E3 Ubiquitin-protein ligase 2 (WWP2) has been found to positively regulate odontoblastic differentiation by monoubiquitinating the transcription factor Kruppel-like factor 5 (KLF5) in a cell culture system. However, the in vivo role of WWP2 in mouse teeth remains unknown. To explore this, here we generated Wwp2 knockout (Wwp2 KO) mice. We found that molars in Wwp2 KO mice exhibited thinner dentin, widened predentin, and reduced numbers of dentinal tubules. In addition, expression of the odontoblast differentiation markers Dspp and Dmp1 was decreased in the odontoblast layers of Wwp2 KO mice. These findings demonstrate that WWP2 may facilitate odontoblast differentiation and dentinogenesis. Furthermore, we show for the first time that phosphatase and tensin homolog (PTEN), a tumor suppressor, is expressed in dental papilla cells and odontoblasts of mouse molars and acts as a negative regulator of odontoblastic differentiation. Further investigation indicated that PTEN is targeted by WWP2 for degradation during odontoblastic differentiation. We demonstrate PTEN physically interacts with and inhibits the transcriptional activity of KLF5 on Dspp and Dmp1. Finally, we found WWP2 was able to suppress the interaction between PTEN and KLF5, which diminished the inhibition effect of PTEN on KLF5. Taken together, this study confirms the essential role of WWP2 and the WWP2-PTEN-KLF5 signaling axis in odontoblast differentiation and dentinogenesis in vivo.

A novel mannose-containing sialoprotein adhesin involved in the binding of Candida albicans cells to DMBT1.

Candida albicans colonizes the oral cavity and causes oral candidiasis and early childhood caries synergistically with cariogenic Streptococcus mutans. Colonization of oral tissues with C. albicans is an essential step in the initiation of these infectious diseases. Deleted in malignant brain tumors 1 (DMBT1), also known as salivary agglutinin or gp-340, belongs to the scavenger receptor cysteine-rich (SRCR) superfamily and has important functions in innate immunity. In the oral cavity, DMBT1 causes microbial adherence to tooth enamel and oral mucosa surfaces, but the adherence of C. albicans to DMBT1 has not been examined. In this study, we investigated the binding of C. albicans to DMBT1 and isolated the fungal components responsible for the binding. Candida albicans specifically bound to DMBT1 and strongly bound to the peptide domain SRCRP2. Binding to SRCRP2 was inhibited by N-acetylneuraminic acid and mannose and by lectins recognizing these sugars. The isolated component had a molecular mass of 25 kDa, contained sialic acid and mannose residues, and inhibited C. albicans binding to SRCRP2. The localization of the 25-kDa protein on the surface of C. albicans cell walls was confirmed by immunostaining and a cell ELISA using an antiserum to the protein, and Western blotting revealed the presence of the 25-kDa protein in the cell wall fraction of C. albicans. These results suggest that the isolated adhesin is localized on the surface of C. albicans cell walls and that sialic acid and mannose residues in the adhesin play a significant role in the binding reaction.

Enteroaggregative Escherichia coli induces altered glycosylation in membrane proteins of cultured human intestinal epithelial cells.

Emerging evidences have suggested that pathogens are capable of manipulating the glycosylation pattern of host-cell glycoconjugates, which may promote their attachment to these cells. Several enteric pathogens are known to induce such altered glycosylation in intestinal epithelium thereby, facilitating the disease process. Enteroaggregative Escherichia coli (EAEC), is one of such pathogens, known to cause acute and persistent diarrhea worldwide. However, glycosylation modulation due to EAEC infection has not been explored so far. In this study, EAEC-induced glycosylation changes in membrane proteins of human small-intestinal and colonic epithelial cell lines were found as revealed by lectin-overlay transblotting using four lectins, among which Sambucus nigra agglutinin (SNA) was selected for subsequent experiments. Several differentially expressed membrane-proteins were detected on SNA-overlay transblots following 2D-PAGE and identified by MALDI-TOF/TOF mass spectrometric analysis. Among these, voltage-dependent anion-selective channel-protein 2 (VDAC2) and prohibitin 2 (PHB2), common to both the cell lines were chosen for further characterization. Reactivity of these proteins to SNA was substantiated by their presence in SNA-agarose affinity chromatography eluted fractions. The plasma membrane localization of VDAC2 and PHB2 in EAEC infected cell lines was validated by confocal microscopy. These proteins were characterized as sialoglycoproteins by SNA-overlay transblots in presence a specific SNA inhibitor i.e., 6'sialyl lactose and deglycosylation using PNGase F, O-glycosidase and neuraminidase. Membrane localization of these sialoglycoproteins was found to facilitate EAEC adherence to human intestinal epithelial cells. SIGNIFICANCE: Our findings regarding EAEC induced altered glycosylation pattern of host cell membrane proteins may help in better understanding of the disease pathogenesis.

A Deadly Embrace: Hemagglutination Mediated by SARS-CoV-2 Spike Protein at Its 22 N-Glycosylation Sites, Red Blood Cell Surface Sialoglycoproteins, and Antibody.

Rouleaux (stacked clumps) of red blood cells (RBCs) observed in the blood of COVID-19 patients in three studies call attention to the properties of several enveloped virus strains dating back to seminal findings of the 1940s. For COVID-19, key such properties are: (1) SARS-CoV-2 binds to RBCs in vitro and also in the blood of COVID-19 patients; (2) although ACE2 is its target for viral fusion and replication, SARS-CoV-2 initially attaches to sialic acid (SA) terminal moieties on host cell membranes via glycans on its spike protein; (3) certain enveloped viruses express hemagglutinin esterase (HE), an enzyme that releases these glycan-mediated bindings to host cells, which is expressed among betacoronaviruses in the common cold strains but not the virulent strains, SARS-CoV, SARS-CoV-2 and MERS. The arrangement and chemical composition of the glycans at the 22 N-glycosylation sites of SARS-CoV-2 spike protein and those at the sialoglycoprotein coating of RBCs allow exploration of specifics as to how virally induced RBC clumping may form. The in vitro and clinical testing of these possibilities can be sharpened by the incorporation of an existing anti-COVID-19 therapeutic that has been found in silico to competitively bind to multiple glycans on SARS-CoV-2 spike protein.

Podocalyxin promotes an impermeable epithelium and inhibits pro-implantation factors to negatively regulate endometrial receptivity.

Embryo implantation is a key step in establishing pregnancy and a major limiting factor in IVF. Implantation requires a receptive endometrium but the mechanisms governing receptivity are not well understood. We have recently discovered that podocalyxin (PCX or PODXL) is a key negative regulator of human endometrial receptivity. PCX is expressed in all endometrial epithelial cells in the non-receptive endometrium but selectively down-regulated in the luminal epithelium at receptivity. We have further demonstrated that this down-regulation is essential for implantation because PCX inhibits embryo attachment and penetration. However, how PCX confers this role is unknown. In this study, through RNAseq analysis of Ishikawa cell line stably overexpressing PCX, we discovered that PCX suppresses expression of genes controlling cell adhesion and communication, but increases those governing epithelial barrier functions, especially the adherens and tight junctions. Moreover, PCX suppresses multiple factors such as LIF and signaling pathways including Wnt and calcium signaling that support receptivity but stimulates anti-implantation genes such as LEFTY2. Functional studies confirmed that PCX promotes epithelial barrier functions by increasing key epithelial junction proteins such as E-cadherin and claudin 4. PCX thus promotes an anti-adhesive and impermeable epithelium while impedes pro-implantation factors to negatively control endometrial receptivity for implantation.

MUC14-Related ncRNA-mRNA Network in Breast Cancer.

Abstract: Background Growing evidences have showed that mucins (MUCs) are linked to occurrence and progression of human cancers. However, a comprehensive study regarding the expression, diagnosis, prognosis and mechanism of MUCs in breast cancer remains absent. Methods: A series of in silico analyses were employed in this study. Results: After performing comprehensive analysis for MUCs, MUC14 was identified as the most potential regulator in breast cancer, with downregulated expression in both mRNA and protein levels and significant diagnostic and prognostic values in breast cancer. Mechanistic exploration revealed that a potential ncRNA-mRNA axis, involving LINC01128/LINC01140/SGMS1-AS1/LINC00667-miR-137/miR-429-BCL2, might be partially responsible for MUC14's functions in breast cancer. Conclusions: Collectively, our study elucidated a key role of MUC14 in breast cancer and also provided some clues for explanation of the molecular action mechanism of MUC14 in breast cancer.

Mouse Dspp frameshift model of human dentinogenesis imperfecta.

Non-syndromic inherited defects of tooth dentin are caused by two classes of dominant negative/gain-of-function mutations in dentin sialophosphoprotein (DSPP): 5' mutations affecting an N-terminal targeting sequence and 3' mutations that shift translation into the - 1 reading frame. DSPP defects cause an overlapping spectrum of phenotypes classified as dentin dysplasia type II and dentinogenesis imperfecta types II and III. Using CRISPR/Cas9, we generated a Dspp-1fs mouse model by introducing a FLAG-tag followed by a single nucleotide deletion that translated 493 extraneous amino acids before termination. Developing incisors and/or molars from this mouse and a DsppP19L mouse were characterized by morphological assessment, bSEM, nanohardness testing, histological analysis, in situ hybridization and immunohistochemistry. DsppP19L dentin contained dentinal tubules but grew slowly and was softer and less mineralized than the wild-type. DsppP19L incisor enamel was softer than normal, while molar enamel showed reduced rod/interrod definition. Dspp-1fs dentin formation was analogous to reparative dentin: it lacked dentinal tubules, contained cellular debris, and was significantly softer and thinner than Dspp+/+ and DsppP19L dentin. The Dspp-1fs incisor enamel appeared normal and was comparable to the wild-type in hardness. We conclude that 5' and 3' Dspp mutations cause dental malformations through different pathological mechanisms and can be regarded as distinct disorders.

SNHG7 promotes the osteo/dentinogenic differentiation ability of human dental pulp stem cells by interacting with hsa-miR-6512-3p in an inflammatory microenvironment.

Excessive inflammation leads to periodontitis, which inhibits the osteogenic differentiation of human dental pulp stem cells (hDPSCs), irreversibly injured and difficultly repaired for the important dental pulp. Hence, it is necessary to study the functional gene to enhance the osteogenic differentiation of hDPSCs. Previous found that SNHG7 expression was increased in the osteogenic differentiation of hDPSCs. However, the regulatory functions of SNHG7 on osteogenic differentiation of hDPSCs in the inflammatory microenvironment still remains unknown. In this study, hDPSCs treatment with 50 ng/mL TNF-α to mimic the inflammatory microenvironment, then cultured in osteoblast differentiation medium for 14 days. SNHG7, miR-6512-3p, BSP, DSPP, DMP-1, RUNX2 and OPN in hDPSCs were detect by RT-qPCR. We found that SNHG7 expression was reduced during the osteogenic differentiation of hDPSCs after different concentrations TNF-α treatment. SNHG7 overexpression improved the TNF-α-induced suppression of calcium deposition, ALP activity, and the expression of BSP, DSPP, DMP-1, RUNX2 and OPN. Furthermore, SNHG7 can sponge with miR-6512-3p. miR-6512-3p expression was increased during the osteogenic differentiation of hDPSCs after different concentrations TNF-α treatment while inhibited after SNHG7 overexpression. knockdown of miR-6512-3p improved the TNF-α-induced suppression of calcium deposition, ALP activity, and the expression of BSP, DSPP, DMP-1, RUNX2 and OPN. Finally, miR-6512-3p overexpression reversed the effect of SNHG7 on the osteo/dentinogenic differentiation of TNF-α-treated hDPSCs. In conclusions, SNHG7 improves the osteogenic differentiation of hDPSCs by inhibiting miR-6512-3p expression under 50 ng/mL TNF-α-induced inflammatory environment, which provided potential targets for the treatment of periodontitis.

EP300/CBP is crucial for cAMP-PKA pathway to alleviate podocyte dedifferentiation via targeting Notch3 signaling.

Podocyte injury is the hallmark of proteinuric glomerular diseases. Notch3 is neo-activated simultaneously in damaged podocytes and podocyte's progenitor cells of FSGS, indicating a unique role of Notch3. We previously showed that activation of cAMP-PKA pathway alleviated podocyte injury possibly via inhibiting Notch3 expression. However, the mechanisms are unknown. In the present study, Notch3 signaling was significantly activated in ADR-induced podocytes in vitro and in PAN nephrosis rats and patients with idiopathic FSGS in vivo, concomitantly with podocyte dedifferentiation. In cultured podocytes, pCPT-cAMP, a selective cAMP-PKA activator, dramatically blocked ADR-induced activation of Notch3 signaling as well as inhibition of cAMP-PKA pathway, thus alleviating the decreased cell viability and podocyte dedifferentiation. Bioinformatics analysis revealed EP300/CBP, a transcriptional co-activator, as a central hub for the crosstalk between these two signaling pathways. Additionally, CREB/KLF15 in cAMP-PKA pathway competed with RBP-J the major transcriptional factor of Notch3 signaling for binding to EP300/CBP. EP300/CBP siRNA significantly inhibited these two signaling transduction pathways and disrupted the interactions between the above major transcriptional factors. These data indicate a crucial role of EP300/CBP in regulating the crosstalk between cAMP-PKA pathway and Notch3 signaling and modulating the phenotypic change of podocytes, and enrich the reno-protective mechanisms of cAMP-PKA pathway.

4-Pyridone-3-carboxylic acid as a benzoic acid bioisostere: Design, synthesis, and evaluation of EP300/CBP histone acetyltransferase inhibitors.

Histone acetyltransferases (HATs) play a crucial role in post-translational modification. Among them, overexpression, mutation, or hyperfunction of EP300/CBP has been associated with various cancers. In this study, we identified the novel compound 2-chloro-5-[5-[(E)-[1-(3-chlorophenyl)-3-methyl-5-oxo-pyrazol-4-ylidene]methyl]-2-furyl]benzoic acid (1) as an EP300 HAT inhibitor via virtual screening. Further research has been focused on the design, synthesis, and in vitro biological evaluation of virtual hit derivatives. The studies revealed that 4-pyridone-3-carboxylic acid derivatives exhibited bioisosterism of benzoic acid. Replacement proved effective, providing compounds with similar EP300 HAT-inhibitory activity and improved cell growth-inhibitory activity compared to the benzoic acid analogs. Through these studies, we identified a potent and selective EP300/CBP HAT inhibitor.

Radiation-Induced Overexpression of TGFß and PODXL Contributes to Colorectal Cancer Cell Radioresistance through Enhanced Motility.

The primary cause of colorectal cancer (CRC) recurrence is increased distant metastasis after radiotherapy, so there is a need for targeted therapeutic approaches to reduce the metastatic-relapse risk. Dysregulation of the cell-surface glycoprotein podocalyxin-like protein (PODXL) plays an important role in promoting cancer-cell motility and is associated with poor prognoses for many malignancy types. We found that CRC cells exposed to radiation demonstrated increased TGFß and PODXL expressions, resulting in increased migration and invasiveness due to increased extracellular matrix deposition. In addition, both TGFß and PODXL were highly expressed in tissue samples from radiotherapy-treated CRC patients compared to those from patients without this treatment. However, it is unclear whether TGFß and PODXL interactions are involved in cancer-progression resistance after radiation exposure in CRC. Here, using CRC cells, we showed that silencing PODXL blocked radiation-induced cell migration and invasiveness. Cell treatment with galunisertib (a TGFß-pathway inhibitor) also led to reduced viability and migration, suggesting that its clinical use may enhance the cytotoxic effects of radiation and lead to the effective inhibition of CRC progression. Overall, the results demonstrate that downregulation of TGFß and its-mediated PODXL may provide potential therapeutic targets for patients with radiotherapy-resistant CRC.

An In Vitro Study of the Effects of Crocin on the Modulation of DSPP, VEGF-A, HLA-G5, STAT3 and CD200 Expression in Human Dental Pulp Stem Cells.

Dental pulp stem cells (DPSCs) have been recommended as promising candidate for cell-based therapeutic applications due to high potentials in tissue repair/regeneration and modulation of immune responses. The gene expression change strategy by natural plant enhancers is an available opportunity to improve the stemness properties of these cells. The objective of this research was the evaluation of Crocin effects (saffron plant's bioactive compound) on immunoregulation and tissue regeneration-related biomarkers expression in human DPSCs. Based on the results of cell viability assay, application of 400 µM and lower concentrations of Crocin had no toxic effects on DPSCs; however, the time-dependent cytotoxic effects were observed at higher concentrations. This study, probably for the first time, detected the surface expression of CD200 in DPSCs with a slight time-dependent upward trend and reported that treatment with Crocin could increase expression of this macromolecule up to many times over. Also, it revealed that this carotenoid significantly led to the time-dependent upregulation of dentin sialophosphoprotein, vascular endothelial growth factor A, human leukocyte antigen-G5, and signal transducer and activator of transcription-3 messenger ribonucleic acids (mRNAs); however, this significant upregulation for STAT3 occurred, followed by a remarkable reduction. The results of this study indicated that cell treatment with Crocin may be effective in improving the stemness capacities of DPSCs. Therefore, the study provided basis for more insights into the biological effects of Crocin on DPSCs that it may aid in the future improvement of mesenchymal stem cell-based therapies.

Endoglycan (PODXL2) is proteolytically processed by ADAM10 (a disintegrin and metalloprotease 10) and controls neurite branching in primary neurons.

Cell adhesion is tightly controlled in multicellular organisms, for example, through proteolytic ectodomain shedding of the adhesion-mediating cell surface transmembrane proteins. In the brain, shedding of cell adhesion proteins is required for nervous system development and function, but the shedding of only a few adhesion proteins has been studied in detail in the mammalian brain. One such adhesion protein is the transmembrane protein endoglycan (PODXL2), which belongs to the CD34-family of highly glycosylated sialomucins. Here, we demonstrate that endoglycan is broadly expressed in the developing mouse brains and is proteolytically shed in vitro in mouse neurons and in vivo in mouse brains. Endoglycan shedding in primary neurons was mediated by the transmembrane protease a disintegrin and metalloprotease 10 (ADAM10), but not by its homolog ADAM17. Functionally, endoglycan deficiency reduced the branching of neurites extending from primary neurons in vitro, whereas deletion of ADAM10 had the opposite effect and increased neurite branching. Taken together, our study discovers a function for endoglycan in neurite branching, establishes endoglycan as an ADAM10 substrate and suggests that ADAM10 cleavage of endoglycan may contribute to neurite branching.

ITGB6-Knockout Suppresses Cholangiocarcinoma Cell Migration and Invasion with Declining PODXL2 Expression.

Intrahepatic cholangiocarcinoma (iCCA) is a heterogeneous bile duct cancer with a poor prognosis. Integrin αvß6 (ß6) has been shown to be upregulated in iCCA and is associated with its subclassification and clinicopathological features. In the present study, two ITGB6-knockout HuCCT1 CCA cell lines (ITGB6-ko cells) were established using the clustered regulatory interspaced short palindromic repeats (CRISPR), an associated nuclease 9 (Cas9) system, and single-cell cloning. RNA sequencing analysis, real-time polymerase chain reaction (PCR), and immunofluorescent methods were applied to explore possible downstream factors. ITGB6-ko cells showed significantly decreased expression of integrin ß6 on flow cytometric analysis. Both cell lines exhibited significant inhibition of cell migration and invasion, decreased wound-healing capability, decreased colony formation ability, and cell cycle dysregulation. RNA sequencing and real-time PCR analysis revealed a remarkable decrease in podocalyxin-like protein 2 (PODXL2) expression in ITGB6-ko cells. Colocalization of PODXL2 and integrin ß6 was also observed. S100 calcium-binding protein P and mucin 1, which are associated with CCA subclassification, were downregulated in ITGB6-ko cells. These results describe the successful generation of ITGB6-ko CCA cell clones with decreased migration and invasion and downregulation of PODXL2, suggesting the utility of integrin ß6 as a possible therapeutic target or diagnostic marker candidate.