Gprc5a is a novel parathyroid hormone-inducible gene and negatively regulates osteoblast proliferation and differentiation.

Teriparatide is a peptide derived from a parathyroid hormone (PTH) and an osteoporosis therapeutic drug with potent bone formation-promoting activity. To identify novel druggable genes that act downstream of PTH signaling and are potentially involved in bone formation, we screened PTH target genes in mouse osteoblast-like MC3T3-E1 cells. Here we show that Gprc5a, encoding an orphan G protein-coupled receptor, is a novel PTH-inducible gene and negatively regulates osteoblast proliferation and differentiation. PTH treatment induced Gprc5a expression in MC3T3-E1 cells, rat osteosarcoma ROS17/2.8 cells, and mouse femurs. Induction of Gprc5a expression by PTH occurred in the absence of protein synthesis and was mediated primarily via the cAMP pathway, suggesting that Gprc5a is a direct target of PTH signaling. Interestingly, Gprc5a expression was induced additively by co-treatment with PTH and 1α, 25-dihydroxyvitamin D3 (calcitriol), or retinoic acid in MC3T3-E1 cells. Reporter analysis of a 1 kb fragment of human GPRC5A promoter revealed that the promoter fragment showed responsiveness to PTH via the cAMP response element, suggesting that GPRC5A is also a PTH-inducible gene in humans. Gprc5a knockdown promoted cell viability and proliferation, as demonstrated by MTT and BrdU assays. Gprc5a knockdown also promoted osteoblast differentiation, as indicated by gene expression analysis and mineralization assay. Mechanistic studies showed that Gprc5a interacted with BMPR1A and suppressed BMP signaling induced by BMP-2 and constitutively active BMP receptors, ALK2 (ACVR1) Q207D and ALK3 (BMPR1A) Q233D. Thus, our results suggest that Gprc5a is a novel gene induced by PTH that acts in an inhibitory manner on both cell proliferation and osteoblast differentiation and is a candidate for drug targets for osteoporosis.

Heterogeneous treatment effect of dose-dense paclitaxel plus carboplatin therapy for advanced ovarian cancer.

A Japanese clinical trial (JGOG3016) showed that dose-dense weekly paclitaxel in combination with carboplatin extensively prolonged overall survival (OS) in patients with advanced ovarian cancer. However, in other clinical trials, dose-dense paclitaxel regimens were not superior to triweekly paclitaxel regimens. In this study, causal tree analysis was applied to explore subpopulations with different treatment effects of dose-dense paclitaxel in a data-driven approach. The 587 participants with stage II-IV ovarian cancer in the JGOG3016 trial were used for model development. The primary endpoint was treatment effect in terms of 3-year OS in patients receiving dose-dense vs. conventional paclitaxel therapies. In patients <50 years, the 3-year OS was similar in both groups; however, it was higher in the dose-dense group in patients ≥50 years. Dose-dense paclitaxel showed strong positive treatment effects in patients ≥50 years with stage II/III disease, BMI <23 kg/m2, non-CC/MC, and residual tumor ≥1 cm. In contrast, although there was no significant difference in OS; the 3-year OS rate was 23% lower in dose-dense paclitaxel than conventional paclitaxel in patients ≥60 years with stage IV cancer. Patients in this group had a particularly lower performance status than other groups. Our causal tree analysis suggested that poor prognosis groups represented by residual tumor tissue ≥1 cm benefit from dose-dense paclitaxel, whereas elderly patients with advanced disease and low-performance status are negatively impacted by dose-dense paclitaxel. These subpopulations will be of interest to future validation studies. Personalized treatments based on clinical features are expected to improve advanced ovarian cancer prognosis.

Increased risk of placenta previa and preterm birth in pregnant women with endometriosis/adenomyosis: A propensity-score matching analysis of a nationwide perinatal database in Japan.

AIM: We aimed to investigate the associations of endometriosis and adenomyosis with pregnancy complications by using a large-scale Japanese database. METHODS: We retrospectively analyzed 145 590 singleton pregnancies from the Japan Perinatal Registry Network Database. Pregnant women registered as having endometriosis or adenomyosis were designated as the case group (EA), whereas the control group (non-EA) was selected using propensity-score matching adjusted for variables such as age, parity, BMI, smoking history, and the use of assisted reproductive technology. The main outcomes included placental malposition, preterm birth, and hypertensive disorders of pregnancy (HDP). RESULTS: In total, 1203 patients from both the EA and non-EA groups were matched and evaluated. The EA group showed significantly higher rates of placenta previa (odds ratio [OR], 3.01; 95% confidence interval [CI], 1.84-4.92), low-lying placenta (OR, 2.02; 95% CI, 1.06-3.86), and preterm birth (OR, 1.44; 95% CI, 1.13-1.84) than the non-EA group. However, no significant difference was observed in the incidence of HDP (OR, 1.22; 95% CI, 0.90-1.66). CONCLUSION: The use of propensity-score matching to analyze a nationwide perinatal database in Japan clarified that EA was associated with increased pregnancy complications, specifically placental malposition, including placenta previa and low-lying placenta, and preterm birth, but not with HDP.

The probiotic Lacticaseibacillus paracasei strain Shirota (LcS) in a fermented milk beverage survives the gastrointestinal tract of generally healthy U.S. Adults.

The probiotic strain Lacticaseibacillus paracasei (previously Lactobacillus casei) strain Shirota (LcS) has demonstrated its survivability in the gastrointestinal tract across populations in different countries. The objective of this study was to validate this survivability in the United States, where evidence is lacking. Faecal samples were collected from 26 healthy individuals (age: 32.0 ± 5.9 years) at baseline, after 7 and 14 days of daily consumption of 80 mL fermented milk containing 108 colony forming units (CFU) LcS/mL, and after a subsequent 14-days of no product consumption. Live LcS counts significantly (p < 0.001) increased after 7 and 14 days of product consumption (6.37 ± 1.18 and 5.24 ± 1.81 log10 CFU/g faeces, respectively) and returned to baseline in 87% of participants. These results indicate LcS survives passage through the gastrointestinal tract of generally healthy U.S. adults, providing support for its uniquely accumulated evidence of universal survival capacity in the gastrointestinal tract.

EHR2CCAS: A framework for mapping EHR to disease knowledge presenting causal chain of disorders - chronic kidney disease example.

OBJECTIVE: The goal of this work was to capture diseases in patients by comprehending the fine-grained medical conditions and disease progression manifested by transitions in medical conditions. We realize this by introducing our earlier work on a state-of-the-art knowledge presentation, which defines a disease as a causal chain of abnormal states (CCAS). Here, we propose a framework, EHR2CCAS, for constructing a system to map electronic health record (EHR) data to CCAS. MATERIALS AND METHODS: EHR2CCAS is a framework consisting of modules that access heterogeneous EHR to estimate the presence of abnormal states in a CCAS for a patient in a given time window. EHR2CCAS applies expert-driven (rule-based) and data-driven (machine learning) methods to identify abnormal states from structured and unstructured EHR data. It features data-driven approaches for unlocking clinical texts and imputations based on the EHR temporal properties and the causal CCAS structure. This study presents the CCAS of chronic kidney disease as an example. A mapping system between the EHR from the University of Tokyo Hospital and CCAS of chronic kidney disease was constructed and evaluated against expert annotation. RESULTS: The system achieved high prediction performance in identifying abnormal states that had strong agreement among annotators. Our handling of narrative varieties in texts and our imputation of the presence of an abnormal state markedly improved the prediction performance. EHR2CCAS presents patient data describing the temporal presence of abnormal states in CCAS, which is useful in individual disease progression management. Further analysis of the differentiation of transition among abnormal states outputted by EHR2CCAS can contribute to detecting disease subtypes. CONCLUSION: This work represents the first step toward combining disease knowledge and EHR to extract abnormality related to a disease defined as fine-grained abnormal states and transitions among them. This can aid in disease progression management and deep phenotyping.

Impaired Myofibroblast Dedifferentiation Contributes to Nonresolving Fibrosis in Aging.

Idiopathic pulmonary fibrosis (IPF) is a fatal age-associated disease with no cure. Although IPF is widely regarded as a disease of aging, the cellular mechanisms that contribute to this age-associated predilection remain elusive. In this study, we sought to evaluate the consequences of senescence on myofibroblast cell fate and fibrotic responses to lung injury in the context of aging. We demonstrated that nonsenescent lung myofibroblasts maintained the capacity for dedifferentiation, whereas senescent/IPF myofibroblasts exhibited an impaired capacity for dedifferentiation. We previously demonstrated that the transcription factor MyoD acts as a critical switch in the differentiation and dedifferentiation of myofibroblasts. Here, we demonstrate that decreased levels of MyoD preceded myofibroblast dedifferentiation and apoptosis susceptibility in nonsenescent cells, whereas MyoD expression remained elevated in senescent/IPF myofibroblasts, which failed to undergo dedifferentiation and demonstrated resistance to apoptosis. Genetic strategies to silence MyoD restored the susceptibility of IPF myofibroblasts to undergo apoptosis and led to a partial reversal of age-associated persistent fibrosis in vivo. The capacity for myofibroblast dedifferentiation and subsequent apoptosis may be critical for normal physiologic responses to tissue injury, whereas restricted dedifferentiation and apoptosis resistance in senescent cells may underlie the progressive nature of age-associated human fibrotic disorders. These studies support the concept that senescence may promote profibrotic effects via impaired myofibroblast dedifferentiation and apoptosis resistance, which contributes to myofibroblast accumulation and ultimately persistent fibrosis in aging.

MUC1 contributes to goblet cell metaplasia and MUC5AC expression in response to cigarette smoke in vivo.

Goblet cell metaplasia (GCM) and mucin overproduction are a hallmark of chronic rhinosinusitis (CRS) and chronic obstructive pulmonary disease (COPD). In the airways, cigarette smoke (CS) induces activation of the epidermal growth factor receptor (EGFR) leading to GCM and overexpression of the gel-forming mucin MUC5AC. Although previous studies have demonstrated that a membrane-bound mucin, MUC1, modulates the activation of CS-induced EGFR, the role of MUC1 in CS-induced GCM and mucin overproduction has not been explored. In response to CS exposure, wild-type (WT) rats displayed Muc1 translocation from the apical surface of airway epithelium to the intracellular compartment of hyperplastic intermediate cells, EGFR phosphorylation, GCM, and Muc5ac overproduction. Similarly, human CRS sinonasal tissues demonstrated hyperplasia of intermediate cells enriched with MUC1 in the intracellular compartment, which was accompanied by GCM and increased MUC5AC expression. To further evaluate the role of Muc1 in vivo, a Muc1 knockout (KO) rat (MUC in humans and Muc in animals) was developed. In contrast to WT littermates, Muc1-KO rats exhibited no activation of EGFR, and were protected from GCM and Muc5ac overproduction. Genetic knockdown of MUC1 in human lung or Muc1 knockout in primary rat airway epithelial cells led to significantly diminished EGF-induced MUC5AC production. Together, these findings suggest that MUC1-dependent EGFR activation mediates CS-induced GCM and mucin overproduction. Strategies designed to suppress MUC1-dependent EGFR activation may provide a novel therapeutic approach for treating mucin hypersecretion in CRS and COPD.

Tumor protein D52 expression is post-transcriptionally regulated by T-cell intercellular antigen (TIA) 1 and TIA-related protein via mRNA stability.

Although tumor protein D52 (TPD52) family proteins were first identified nearly 20 years ago, their molecular regulatory mechanisms remain unclear. Therefore, we investigated the post-transcriptional regulation of TPD52 family genes. An RNA immunoprecipitation (RIP) assay showed the potential binding ability of TPD52 family mRNAs to several RNA-binding proteins, and an RNA degradation assay revealed that TPD52 is subject to more prominent post-transcriptional regulation than are TPD53 and TPD54. We subsequently focused on the 3'-untranslated region (3'-UTR) of TPD52 as a cis-acting element in post-transcriptional gene regulation. Several deletion mutants of the 3'-UTR of TPD52 mRNA were constructed and ligated to the 3'-end of a reporter green fluorescence protein gene. An RNA degradation assay revealed that a minimal cis-acting region, located in the 78-280 region of the 5'-proximal region of the 3'-UTR, stabilized the reporter mRNA. Biotin pull-down and RIP assays revealed specific binding of the region to T-cell intracellular antigen 1 (TIA-1) and TIA-1-related protein (TIAR). Knockdown of TIA-1/TIAR decreased not only the expression, but also the stability of TPD52 mRNA; it also decreased the expression and stability of the reporter gene ligated to the 3'-end of the 78-280 fragment. Stimulation of transforming growth factor-ß and epidermal growth factor decreased the binding ability of these factors, resulting in decreased mRNA stability. These results indicate that the 78-280 fragment and TIA-1/TIAR concordantly contribute to mRNA stability as a cis-acting element and trans-acting factor(s), respectively. Thus, we here report the specific interactions between these elements in the post-transcriptional regulation of the TPD52 gene.

Muc1 knockout potentiates murine lung carcinogenesis involving an epiregulin-mediated EGFR activation feedback loop.

Mucin 1 (MUC1) is a tumor antigen that is aberrantly overexpressed in various cancers, including lung cancer. Our previous in vitro studies showed that MUC1 facilitates carcinogen-induced EGFR activation and transformation in human lung bronchial epithelial cells (HBECs), which along with other reports suggests an oncogenic property for MUC1 in lung cancer. However, direct evidence for the role of MUC1 in lung carcinogenesis is lacking. In this study, we used the 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced A/J mouse lung tumor model to investigate the effect of whole-body Muc1 knockout (KO) on carcinogen-induced lung carcinogenesis. Surprisingly, lung tumor multiplicity was significantly increased in Muc1 KO compared to wild-type (WT) mice. The EGFR/AKT pathway was unexpectedly activated, and expression of the EGFR ligand epiregulin (EREG) was increased in the lung tissues of the Muc1 KO compared to the WT mice. EREG stimulated proliferation and protected against cigarette smoke extract (CSE)-induced cytotoxicity in in vitro cultured human bronchial epithelial cells. Additionally, we determined that MUC1 was expressed in human fibroblast cell lines where it suppressed CSE-induced EREG production. Further, suppression of MUC1 cellular activity with GO-201 enhanced EREG production in lung cancer cells, which in turn protected cancer cells from GO-201-induced cell death. Moreover, an inverse association between MUC1 and EREG was detected in human lung cancer, and EREG expression was inversely associated with patient survival. Together, these results support a promiscuous role of MUC1 in lung cancer development that may be related to cell-type specific functions of MUC1 in the tumor microenvironment, and MUC1 deficiency in fibroblasts and malignant cells results in increased EREG production that activates the EGFR pathway for lung carcinogenesis.

Muc1 deficiency exacerbates pulmonary fibrosis in a mouse model of silicosis.

BACKGROUND: MUC1 (MUC in human and Muc in animals) is a membrane-tethered mucin expressed on the apical surface of lung epithelial cells. However, in the lungs of patients with interstitial lung disease, MUC1 is aberrantly expressed in hyperplastic alveolar type II epithelial (ATII) cells and alveolar macrophages (AM), and elevated levels of extracellular MUC1 are found in bronchoalveolar lavage (BAL) fluid and the serum of these patients. While pro-fibrotic effects of extracellular MUC1 have recently been described in cultured fibroblasts, the contribution of MUC1 to the pathobiology of pulmonary fibrosis is unknown. In this study, we hypothesized that MUC1 deficiency would reduce susceptibility to pulmonary fibrosis in a mouse model of silicosis. METHODS: We employed human MUC1 transgenic mice, Muc1 deficient mice and wild-type mice on C57BL/6 background in these studies. Some mice received a one-time dose of crystalline silica instilled into their oropharynx in order to induce pulmonary fibrosis and assess the effects of Muc1 deficiency on fibrotic and inflammatory responses in the lung. RESULTS: As previously described in other mouse models of pulmonary fibrosis, we found that extracellular MUC1 levels were markedly increased in whole lung tissues, BALF and serum of human MUC1 transgenic mice after silica. We also detected an increase in total MUC1 levels in the lungs of these mice, indicating that production as well as release contributed to elevated levels after lung injury. Immunohistochemical staining revealed that increased MUC1 expression was mostly confined to ATII cells and AMs in areas of fibrotic remodeling, illustrating a pattern similar to the expression of MUC1 in human fibrotic lung tissues. However, contrary to our hypothesis, we found that Muc1 deficiency resulted in a worsening of fibrotic remodeling in the mouse lung as judged by an increase in number of silicotic nodules, an increase in lung collagen deposition and an increase in the severity of pulmonary inflammation. CONCLUSIONS: Altogether, our results indicate that Muc1 has anti-fibrotic properties in the mouse lung and suggest that elevated levels of MUC1 in patients with interstitial lung disease may serve a protective role, which aims to limit the severity of tissue remodeling in the lung.

Pseudomonas aeruginosa increases MUC1 expression in macrophages through the TLR4-p38 pathway.

Alveolar macrophages (AMs) play a critical role in the clearance of Pseudomonas aeruginosa (Pa) from the airways. However, hyper-activation of macrophages can impair bacterial clearance and contribute to morbidity and mortality. MUC1 mucin is a membrane-tethered, high molecular mass glycoprotein expressed on the apical surface of mucosal epithelial cells and some hematopoietic cells, including macrophages, where it counter-regulates inflammation. We recently reported that Pa up-regulates the expression of MUC1 in primary human AMs and THP-1 macrophages, and that increased MUC1 expression in these cells prevents hyper-activation of macrophages that appears to be important for host defense against severe pathology of Pa lung infection. The aims of this study were to elucidate the mechanism by which Pa increases MUC1 expression in macrophages. The results showed that: (a) Pa stimulation of THP-1 macrophages increased MUC1 expression both at transcriptional and protein levels in a dose-dependent manner; (b) Both Pa- and LPS-induced MUC1 expression in THP-1 cells were significantly diminished by an inhibitory peptide of TLR4; and (c) LPS-stimulated MUC1 expression was diminished at both the mRNA and protein levels by an inhibitor of the p38 mitogen-activated protein kinase, but not by inhibitors of ERK1/2, JNK, or IKK. We conclude that Pa-stimulated MUC1 expression in THP-1 macrophages is regulated mainly through the TLR4-p38 signaling pathway.

Membrane-Tethered MUC1 Mucin Counter-Regulates the Phagocytic Activity of Macrophages.

MUC1 (MUC in human; Muc in animals) is a transmembrane mucin glycoprotein expressed in mucosal epithelial cells and hematopoietic cells. MUC1 is involved in the resolution of inflammation during airway Pseudomonas aeruginosa (Pa) infection by suppressing Toll-like receptor signaling in airway epithelial cells. Although alveolar macrophages are recognized as critical mediators of cell-mediated immunity against microorganisms inhaled into the airways, the role of MUC1 in regulating their response is unknown. The aims of this study were to determine whether macrophages express MUC1, and, if so, whether MUC1 expression might be associated with macrophage M0/M1/M2 differentiation or phagocytic activity. Human and mouse MUC1/Muc1 expression was drastically up-regulated in classically activated (M1) macrophages compared with nonactivated (M0) and alternatively activated (M2) macrophages. M1 polarization and Pa stimulation each increased MUC1 ectodomain shedding from the macrophage surface in a TNF-α-converting enzyme-dependent manner. MUC1/Muc1 deficiency in M0 macrophages increased adhesion and phagocytosis of Pa and Escherichia coli compared with MUC1/Muc1-expressing cells, and attenuation of phagocytosis by MUC1 was augmented after polarization into M1 macrophages compared with M0 macrophages. Finally, MUC1/Muc1 deficiency in macrophages increased reactive oxygen species production and TNF-α release in response to Pa compared with MUC1/Muc1-sufficient cells. These results indicate that MUC1/Muc1 expression by macrophages is predominantly in the M1 subtype, and that MUC1/Muc1 expression in these cells decreases their phagocytic activity in an antiinflammatory manner.

Pseudomonas aeruginosa stimulates tyrosine phosphorylation of and TLR5 association with the MUC1 cytoplasmic tail through EGFR activation.

BACKGROUND: MUC1 is a membrane-tethered mucin expressed on the surface of epithelial and hematopoietic cells. Previous studies have established that MUC1 attenuates airway inflammation in response to Pseudomonas aeruginosa (Pa) through suppression of Toll-like receptor (TLR) signaling. Here, we elucidate the mechanism through which the MUC1 cytoplasmic tail (CT) inhibits TLR5 signaling in response to Pa and its flagellin in primary normal human bronchial epithelial (NHBE) cells. METHODS: NHBE and human and mouse macrophages were stimulated with Pa or flagellin and transforming growth factor-α (TGF-α) and tumor necrosis factor-α (TNF-α) levels in cell culture supernatants were measured by ELISA. NHBE cells were stimulated with Pa, flagellin, or TNF-α and MUC1-CT, and epidermal growth factor receptor (EGFR) levels were measured by immunoblotting. NHBE cells were stimulated with Pa and MUC1-CT/TLR5 and MUC1-CT/EGFR association were detected by co-immunoprecipitation. RESULTS: Stimulation of NHBE cells with Pa and flagellin each increased release of the EGFR ligand, TGF-α, from NHBE cells. Both stimuli also activated EGFR tyrosine phosphorylation in these same cells. By contrast, stimulation of NHBE cells with Pa failed to induce TNF-α release, whereas stimulation of human or mouse macrophages with Pa promoted TNF-α release. Stimulation of NHBE cells with recombinant TNF-α increased both MUC1 and EGFR protein levels, and stimulation of these cells with Pa enhanced MUC1-CT tyrosine phosphorylation and increased MUC1-CT/TLR5 and MUC1-CT/EGFR protein association, in an EGFR-dependent manner. CONCLUSIONS: These results indicate that in response to Pa or flagellin, EGFR associates with and tyrosine phosphorylates MUC1-CT in primary NHBE cells, leading to increased MUC1-CT association with TLR5. Based on prior studies in tumor cells, increased MUC1-CT/TLR5 association in NHBE cells is predicted to competitively inhibit Pa/flagellin-stimulated TLR5 activation, reduce TLR5-dependent cell signaling, and down-regulate airway inflammation. Given that MUC1 is a universal suppressor of TLR signaling, the results from this study suggest that abnormal interactions between MUC1 and EGFR or TLRs may lead to the development of chronic inflammatory diseases. Thus, this is an important finding from the clinical point of view.

Lipopolysaccharide decreases single immunoglobulin interleukin-1 receptor-related molecule (SIGIRR) expression by suppressing specificity protein 1 (Sp1) via the Toll-like receptor 4 (TLR4)-p38 pathway in monocytes and neutrophils.

Single immunoglobulin interleukin-1 receptor-related molecule (SIGIRR) is one of the immunoglobulin-like membrane proteins that is crucial for negative regulation of toll-like receptor 4 (TLR4) and interleukin-1 receptor. Despite the importance of understanding its expression and function, knowledge is limited on the regulatory mechanism in the epithelial tissues, such as the liver, lung, and gut, where its predominant expression is originally described. Here, we found expression of SIGIRR in non-epithelial innate immune cells, including primary peripheral blood monocytes, polymorphonuclear neutrophils, monocytic RAW264 cells, and neutrophilic-differentiated HL-60 cells. Consistent with previous findings in epithelial tissues, SIGIRR gene and protein expression were also down-regulated by LPS treatment in a time-dependent manner in primary blood monocytes and polymorphonuclear neutrophils. A reduction was also observed in RAW264 and differentiated HL-60 cells. Notably, exogenous introduction of the dominant negative form of TLR4 and siRNA of p38 resulted in inhibition of LPS-induced SIGIRR down-regulation, whereas treatment with p38 activator anisomycin showed a dose-dependent decrease in SIGIRR expression, suggesting TLR4-p38 signal as a critical pathway for LPS-induced SIGIRR down-regulation. Finally, reporter gene and chromatin immunoprecipitation assays demonstrated that Sp1 is a key factor that directly binds to the proximal promoter of SIGIRR gene and consequently regulates basal SIGIRR expression, which is negatively regulated by the LPS-dependent TLR4-p38 pathway. In summary, the data precisely demonstrate how LPS down-regulates SIGIRR expression and provide a role of LPS signal that counteracts Sp1-dependent basal promoter activation of SIGIRR gene via TLR4-p38 pathway in non-epithelial innate immune cells.

MUC1 regulates epithelial inflammation and apoptosis by PolyI:C through inhibition of Toll/IL-1 receptor-domain-containing adapter-inducing IFN-ß (TRIF) recruitment to Toll-like receptor 3.

MUC1/Muc1 (MUC1 in humans, Muc1 in animals) is a membrane-tethered mucin expressed by airway epithelial cells and plays an antiinflammatory role during airway bacterial infection. We previously demonstrated that MUC1/Muc1 is a negative regulator of Toll-like receptor (TLR) inflammatory signaling mediated through the myeloid differentiation primary response gene 88 (MyD88) adaptor protein. In the present study, we determined whether MUC1 regulates MyD88-independent TLR signaling mediated through the TLR3-Toll/IL-1 receptor-domain-containing adapter-inducing IFN-ß (TRIF) pathway in response to poly(I:C). Compared with MUC1/Muc1-expressing controls, cells deficient in MUC1/Muc1 were more prone to poly(I:C)-induced apoptosis; had increased poly(I:C)-driven activation of caspase-3, caspase-8, IFN regulatory factor-3, and NF-κB; and displayed heightened IFN-ß gene expression. MUC1 overexpression by these cells had the opposite effects. Reciprocal coimmunoprecipitation experiments established constitutive TLR3/MUC1-CT (cytoplasmic tail) protein interaction in human embryonic kidney (HEK)293T cells overexpressing the two proteins and in lung epithelial cells expressing the endogenous proteins, the latter of which was confirmed by immunofluorescence colocalization of TLR3 with MUC1-CT. Coimmunoprecipitation studies also revealed that MUC1 overexpression by HEK293T cells reduced poly(I:C)-induced TLR3/TRIF protein interaction. Finally, MUC1 overexpression had no effect on TRIF-dependent auto-activation of TLR3 signaling, suggesting that the site of action of the MUC1-CT in TLR3 signaling is not downstream of TRIF. These data indicate that MUC1-CT counter-regulates apoptotic and inflammatory responses of airway epithelial cell through constitutive association with TLR3, thereby inhibiting poly(I:C)-induced recruitment of TRIF to TLR3.

A signaling pathway consisting of miR-551b, catalase and MUC1 contributes to acquired apoptosis resistance and chemoresistance.

Acquired chemoresistance is a major challenge in cancer therapy. While the oncoprotein Mucin-1 (MUC1) performs multiple roles in the development of diverse human tumors, whether MUC1 is involved in acquired chemoresistance has not been determined. Using an acquired chemoresistance lung cancer cell model, we show that MUC1 expression was substantially increased in cells with acquired apoptosis resistance (AR). Knockdown of MUC1 expression effectively increased the sensitivity of these cells to the apoptotic cytotoxicity of anticancer therapeutics, suggesting that MUC1 contributes to acquired chemoresistance. Decreased catalase expression and increased cellular reactive oxygen species (ROS) accumulation were found to be associated with MUC1 overexpression. Scavenging ROS with butylated hydroxyanisole or supplying exogenous catalase dramatically suppressed MUC1 expression through destabilizing MUC1 protein, suggesting that reduced catalase expression mediated ROS accumulation is accounted for MUC1 overexpression. Further, we found that increased miR-551b expression in the AR cells inhibited the expression of catalase and potentiated ROS accumulation and MUC1 expression. Finally, by manipulating MUC1 expression, we found that MUC1 promotes EGFR-mediated activation of the cell survival cascade involving Akt/c-FLIP/COX-2 in order to protect cancer cells from responding to anticancer agents. Thus, our results establish a pathway consisting of miR-551b/catalase/ROS that results in MUC1 overexpression, and intervention against this pathway could be exploited to overcome acquired chemoresistance.

Membrane-tethered MUC1 mucin is phosphorylated by epidermal growth factor receptor in airway epithelial cells and associates with TLR5 to inhibit recruitment of MyD88.

MUC1 is a membrane-tethered mucin glycoprotein expressed on the apical surface of mucosal epithelial cells. Previous in vivo and in vitro studies established that MUC1 counterregulates airway inflammation by suppressing TLR signaling. In this article, we elucidate the mechanism by which MUC1 inhibits TLR5 signaling. Overexpression of MUC1 in HEK293 cells dramatically reduced Pseudomonas aeruginosa-stimulated IL-8 expression and decreased the activation of NF-κB and MAPK compared with cells not expressing MUC1. However, overexpression of MUC1 in HEK293 cells did not affect NF-κB or MAPK activation in response to TNF-α. Overexpression of MyD88 abrogated the ability of MUC1 to inhibit NF-κB activation, and MUC1 overexpression inhibited flagellin-induced association of TLR5/MyD88 compared with controls. The MUC1 cytoplasmic tail associated with TLR5 in all cells tested, including HEK293T cells, human lung adenocarcinoma cell line A549 cells, and human and mouse primary airway epithelial cells. Activation of epidermal growth factor receptor tyrosine kinase with TGF-α induced phosphorylation of the MUC1 cytoplasmic tail at the Y46EKV sequence and increased association of MUC1/TLR5. Finally, in vivo experiments demonstrated increased immunofluorescence colocalization of Muc1/TLR5 and Muc1/phosphotyrosine staining patterns in mouse airway epithelium and increased Muc1 tyrosine phosphorylation in mouse lung homogenates following P. aeruginosa infection. In conclusion, epidermal growth factor receptor tyrosine phosphorylates MUC1, leading to an increase in its association with TLR5, thereby competitively and reversibly inhibiting recruitment of MyD88 to TLR5 and downstream signaling events. This unique ability of MUC1 to control TLR5 signaling suggests its potential role in the pathogenesis of chronic inflammatory lung diseases.

Cell wall polysaccharide enhances Lacticaseibacillus paracasei strain Shirota growth in milk and contributes to acid and bile tolerance.

LCPS-1, a cell wall polysaccharide (CWPS), is bound to the cell wall of the probiotic Lacticaseibacillus paracasei (formerly known as Lactobacillus casei) strain Shirota (LcS). Generally, the role of CWPS in the viability and survivability of bacteria is yet to be fully understood. This study aimed to elucidate the role of LCPS-1 in the viability and survivability of LcS. A mutant strain completely lacking LCPS-1 was constructed and evaluated for growth in bovine and soy milk and susceptibility to acid and bile. The growth of the mutant in bovine and soy milk temporarily stalled after the late logarithmic phase while wild-type LcS continued growing, resulting in a significantly lower number of viable cells for the mutant strain (p < 0.01). Significantly higher cell death relative to that of the wild-type strain was observed for the mutant strain following acid treatment at pH 3.0 (p < 0.01), with 60 and 92 % survival, respectively. The absence of LCPS-1 also reduced the survival rate of LcS cells from 3.3 to 0.8 % following 0.2 % bile treatment. The survival rate of the mutant after consecutive treatment with acid and bile was 19 %, while 73 % of the wild-type LcS survived. These results indicate that LCPS-1 leads to higher LcS growth in milk and improves tolerance to acid and bile. This study reveals the contribution of probiotic bacterial CWPS to acidic and gastrointestinal stress tolerance. Based on these findings, characterizing and modifying CWPS in probiotic strains could enhance manufacturing yields and improve gastrointestinal stress tolerance after consumption by hosts, ultimately advancing the development of more effective probiotics.