Salinomycin alleviates osteoarthritis progression via inhibiting Wnt/ß-catenin signaling.

Osteoarthritis (OA) is the most prevalent degenerative whole-joint disease characterized by cartilage degeneration, synovial hyperplasia, osteophyte formation, and subchondral bone sclerosis. Currently there are no disease-modifying treatments available for OA because its etiology and pathogenesis are largely unknown. Here we report that a natural carboxylic polyether ionophore that is used as an anti-tumor drug, salinomycin (SAL), may be a promising therapeutic drug for OA in the future. We found that SAL showed no cytotoxicity on mouse chondrocytes and displayed a protective effect against interleukin-1ß (IL-1ß), in cultured mouse chondrocytes and cartilage explants. Treatment with low SAL concentrations directly upregulated the anabolism factors collagen II and aggrecan, while it inhibited the catabolic factors matrix metalloproteinase-13 (MMP13) and metalloproteinase with thrombospondin motifs-5 (ADAMTS5) to protect against extracellular matrix (ECM) degradation, and also suppressed inflammatory responses in mouse chondrocytes. Furthermore, SAL reduced the severity of OA-associated changes and delayed cartilage destruction, subchondral bone sclerosis, and osteophyte formation in a destabilized medial meniscus (DMM) surgery-induced mouse OA model. Mechanistically, a low SAL concentration induced anabolism and inhibited catabolism in chondrocytes via inhibiting Lrp6 phosphorylation and Wnt/ß-catenin signaling. Our results suggested that SAL may serve as a potential disease-modifying therapeutic against OA pathogenesis.

Polydatin inhibits IL-1ß-mediated chondrocyte inflammation and ameliorates cartilage degradation: Involvement of the NF-κB and Wnt/ß-catenin pathways.

Osteoarthritis (OA) is a highly prevalent chronic joint disease that involves extracellular matrix (ECM) degradation and articular cartilage inflammation. Polydatin (PD) can alleviate inflammatory reactions in numerous diseases. The present study aimed to investigate the chondroprotective and anti-inflammatory effects of PD on interleukin (IL)- 1ß-treated chondrocytes in vitro and anterior cruciate ligament transection-induced rat OA models in vivo. Primary chondrocytes were isolated from SD rats and cultured. Only second-passage cells were used for subsequent experiments. Counting kit-8, quantitative real-time polymerase chain reaction, western blotting, enzyme-linked immunosorbent assay, and immunofluorescence were used to detect relevant indices. Rat OA models were established to obtain in vivo data. PD treatment decreased the production of nitric oxide (NO), prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), and IL-6 during IL-1ß-stimulated chondrocyte inflammation. Moreover, PD upregulated aggrecan and collagen II expression, whereas downregulated a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) and matrix metalloproteinase-13 (MMP-13) expression on IL-1ß-mediated chondrocytes. Additionally, PD reduced IL-1ß-stimulated NF-κB and Wnt/ß-catenin activation and nuclear translocation. The results of histological analysis and scoring revealed that OA in the rat models was effectively ameliorated by the intra-articular injection of PD. PD suppressed IL-1ß-stimulated iNOS, COX-2, NO, and PGE2 production, TNF-α, IL-6, collagen X, MMP-13, and ADAMTS-5 expression, collagen II and aggrecan degeneration by inhibiting NF-κB and Wnt/ß-catenin signaling in vitro. PD also mitigated OA progression in the rat models, thereby providing reliable data that PD could serve as a promising candidate for OA therapy.

Circular RNA circ_0020014 contributes to osteoarthritis progression via miR-613/ADAMTS5 axis.

Circular RNAs (circRNAs) have been shown to be significant regulators in osteoarthritis (OA), whereas the functional effect of circ_0020014 in OA remains unclear. Our goal was to try and understand the underlying regulatory mechanism of circ_0020014 in OA. The cartilage tissue was obtained from OA patients and trauma patients. Interleukin-1ß (IL-1ß)-treated chondrocytes (CHON-001) were used as the in vitro cellular model for OA. The expression levels of circ_0020014, microRNA-613 (miR-613), and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) were examined by real-time quantitative polymerase chain reaction (RT-qPCR). The protein level was detected using the western blot assay. Cell viability and apoptosis were measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazol-3-ium bromide (MTT) and flow cytometry assays, respectively. The secretion of inflammatory cytokine was determined by enzyme-linked immunosorbent assay (ELISA). Circ_0020014 was upregulated in OA cartilage tissues and IL-1ß-treated CHON-001 cells, compared with that in healthy cartilage tissues and untreated cells. IL-1ß treatment induced cell injury by promoting inflammation and apoptosis, and inhibiting cell viability and extracellular matrix (ECM) accumulation in chondrocytes. Circ_0020014 knockdown significantly protected CHON-001 cells from IL-1ß-induced cell dysfunction. MiR-613 was targeted by circ_0020014 and negatively regulated ADAMTS5 expression. In addition, miR-613 downregulation or ADAMTS5 overexpression partly lessened the protective effect of circ_0020014 knockdown on IL-1ß-treated CHON-001 cells. Collectively, circ_0020014 acted as a miR-613 sponge to regulate ADAMTS5 expression, thereby protecting chondrocytes from IL-1ß-induced inflammatory damage, which might be a novel diagnostic marker for OA.

Estradiol and RSPO3 regulate vertebral trabecular bone mass independent of each other.

Osteoporosis is an age-dependent serious skeletal disease that leads to great suffering for the patient and high social costs, especially as the global population reaches higher age. Decreasing estrogen levels after menopause result in a substantial bone loss and increased fracture risk, whereas estrogen treatment improves bone mass in women. RSPO3, a secreted protein that modulates WNT signaling, increases trabecular bone mass and strength in the vertebrae of mice, and is associated with trabecular density and risk of distal forearm fractures in humans. The aim of the present study was to determine if RSPO3 is involved in the bone-sparing effect of estrogens. We first observed that estradiol (E2) treatment increases RSPO3 expression in bone of ovariectomized (OVX) mice, supporting a possible role of RSPO3 in the bone-sparing effect of estrogens. As RSPO3 is mainly expressed by osteoblasts in the bone, we used a mouse model devoid of osteoblast-derived RSPO3 (Runx2-creRspo3flox/flox mice) to determine if RSPO3 is required for the bone-sparing effect of E2 in OVX mice. We confirmed that osteoblast-specific RSPO3 inactivation results in a substantial reduction in trabecular bone mass and strength in the vertebrae. However, E2 increased vertebral trabecular bone mass and strength similarly in mice devoid of osteoblast-derived RSPO3 and control mice. Unexpectedly, osteoblast-derived RSPO3 was needed for the full estrogenic response on cortical bone thickness. In conclusion, although osteoblast-derived RSPO3 is a crucial regulator of vertebral trabecular bone, it is required for a full estrogenic effect on cortical, but not trabecular, bone in OVX mice. Thus, estradiol and RSPO3 regulate vertebral trabecular bone mass independent of each other.NEW & NOTEWORTHY Osteoblast-derived RSPO3 is known to be a crucial regulator of vertebral trabecular bone. Our new findings show that RSPO3 and estrogen regulate trabecular bone independent of each other, but that RSPO3 is necessary for a complete estrogenic effect on cortical bone.

FSH promotes the proliferation of sheep granulosa cells by inhibiting the expression of TSP1.

Thrombospondin (TSP1) plays an important role as an antiangiogenic factor in the reproductive system of female mammals. However, its expression and function in sheep are still unclear. In the present research, the Altay sheep (a native Chinese breed) was used to analyze the expression of TSP1 in the ovary and its potential function in granulosa cells. TSP1 was widely expressed in most tissues, as shown by qPCR. In the ovary, TSP1 mRNA expression decreased during follicular to luteal growth. The TSP1 protein was expressed in a wide variety of follicles of different diameters and localized to the cytoplasm and nucleus of granulosa cells. In in vitro studies, follicle-stimulating hormone (FSH) significantly inhibited the expression of TSP1 in sheep granulosa cells. Functionally, FSH- and TSP1-specific siRNAs can promote the proliferation of sheep granulosa cells. In contrast, TSP1 mimetic peptide, ABT510, offsets the proliferation of sheep granulosa cells. Different signaling pathway inhibitors all promoted FSH-inhibited TSP1 expression, but each inhibitor had different effects on TSP1. Among them, the PI3K and ERK pathway inhibitors significantly promoted TSP1 expression and inhibited the proliferation of sheep granulosa cells.

Thrombospondin 1 and 2 along with PEDF inhibit angiogenesis and promote lymphangiogenesis in intrahepatic cholangiocarcinoma.

BACKGROUND & AIMS: The microenvironment of intrahepatic cholangiocarcinoma (iCCA) is hypovascularized, with an extensive lymphatic network. This leads to rapid cancer spread into regional lymph nodes and the liver parenchyma, precluding curative treatments. Herein, we investigated which factors released in the iCCA stroma drive the inhibition of angiogenesis and promote lymphangiogenesis. METHODS: Quantitative proteomics was performed on extracellular fluid (ECF) proteins extracted both from cancerous and non-cancerous tissues (NCT) of patients with iCCA. Computational biology was applied on a proteomic dataset to identify proteins involved in the regulation of vessel formation. Endothelial cells incubated with ECF from either iCCA or NCT specimens were used to assess the role of candidate proteins in 3D vascular assembly, cell migration, proliferation and viability. Angiogenesis and lymphangiogenesis were further investigated in vivo by a heterotopic transplantation of bone marrow stromal cells, along with endothelial cells in SCID/beige mice. RESULTS: Functional analysis of upregulated proteins in iCCA unveils a soluble angio-inhibitory milieu made up of thrombospondin (THBS)1, THBS2 and pigment epithelium-derived factor (PEDF). iCCA ECF was able to inhibit in vitro vessel morphogenesis and viability. Antibodies blocking THBS1, THBS2 and PEDF restored tube formation and endothelial cell viability to levels observed in NCT ECF. Moreover, in transplanted mice, the inhibition of blood vessel formation, the de novo generation of the lymphatic network and the dissemination of iCCA cells in lymph nodes were shown to depend on THBS1, THBS2 and PEDF expression. CONCLUSIONS: THBS1, THBS2 and PEDF reduce blood vessel formation and promote tumor-associated lymphangiogenesis in iCCA. Our results identify new potential targets for interventions to counteract the dissemination process in iCCA. LAY SUMMARY: Intrahepatic cholangiocarcinoma is a highly aggressive cancer arising from epithelial cells lining the biliary tree, characterized by dissemination into the liver parenchyma via lymphatic vessels. Herein, we show that the proteins THBS1, THBS2 and PEDF, once released in the tumor microenvironment, inhibit vascular growth, while promoting cancer-associated lymphangiogenesis. Therefore, targeting THBS1, THBS2 and PEDF may be a promising strategy to reduce cancer-associated lymphangiogenesis and counteract the invasiveness of intrahepatic cholangiocarcinoma.

Thrombospondin-5 and fluvastatin promote angiogenesis and are protective against endothelial cell apoptosis.

The thrombospondins (TSPs), multifunctional matricellular proteins, are known mediators of endothelial cell (EC) angiogenesis and apoptosis. TSP-1, an antiangiogenic molecule, is important in the progression of vascular disease, in part by inducing EC apoptosis. TSP-2, although less studied, also induces EC apoptosis and inhibits angiogenesis. The effects of TSP-5 are largely unexplored in ECs, but TSP-5 is believed to be protective against arterial disease. Statin drugs have been shown to have beneficial pleiotropic effects, including decreasing EC apoptosis, increasing angiogenesis, and blocking TSP signaling. We hypothesized TSP-5 will be proangiogenic and antiapoptotic, and statin pretreatment would reverse the proapoptotic and antiangiogenic phenotype of TSP-1 and TSP-2. ECs were exposed to serum-free medium, TSP-1, TSP-2, or TSP-5 with or without fluvastatin pretreatment. Quantitative real-time polymerase chain reaction was performed on 96 apoptosis and 96 angiogenesis-related genes using microfluidic card assays. Angiogenesis was measured using Matrigel assays, while apoptosis was measured by fluorescent caspase assay. TSP-5 suppressed apoptotic genes and had a mixed effect on the angiogenic genes; however, TSP-5 did not alter apoptois but was proangiogenic. Pretreatment with fluvastatin downregulated proapoptotic genes and apoptosis and upregulated proangiogenic genes and angiogenesis. Findings indicate TSP-5 and fluvastatin have a protective effect on ECs, being proangiogenic and reversing the antiangiogenic effects of TSP-1 and TSP-2. In conclusion, TSP-5 and fluvastatin may be beneficial for inducing angiogenesis in the setting of ischemia.

Thrombospondin-4 induces prolongation of action potential duration in rat isolated ventricular myocytes.

Expression of thrombospondin-4 (TSP-4), a matricellular protein, is increased in the heart tissue of various cardiac disease models. In dorsal root ganglion neurons, TSP-4 inhibits L-type Ca2+ channel (LTCC) activity. Although TSP-4 might be related to the electrophysiological properties in heart, it remains to be clarified. The present study aimed to clarify the effects of TSP-4 on action potential (AP), LTCC current (ICaL) and voltage-dependent K+ (Kv) channel current (IKv) in rat isolated ventricular myocytes by a patch clamp technique. Ventricular myocytes were isolated from the heart of adult male Wistar rats. The ventricular myocytes were treated with TSP-4 (5 nM) or its vehicle for 4 hr. Then, whole-cell patch clamp technique was performed to measure AP (current-clamp mode) and ICaL and IKv (voltage-clamp mode). The mRNA expression of Kv channels was examined by reverse transcription-polymerase chain reaction. TSP-4 had no effect on the resting membrane potential and peak amplitude of AP. On the other hand, TSP-4 significantly prolonged AP duration (APD) at 50% and 90% repolarization. TSP-4 significantly inhibited the peak amplitudes of ICaL and IKv. TSP-4 had no effect on mRNA expression of Kv channels (Kcna4, Kcna5, Kcnb1, Kcnd2 and Kcnd3). The present study for the first time demonstrated that TSP-4 prolongs APD in rat ventricular myocytes, which is possibly mediated through the suppression of Kv channel activity.

MiR-106a aggravates sepsis-induced acute kidney injury by targeting THBS2 in mice model.

PURPOSE: To investigate the role and related mechanisms of miR-106a in sepsis-induced AKI. METHODS: Serum from sepsis and healthy patients was collected, sepsis mouse model was established by cecal ligation and puncture (CLP). TCMK-1 cells were treated with lipopolysaccharide (LPS) and transfected with THBS2-small interfering RNA (siTHBS2), miR-106a inhibitor, miR-106a mimics and their negative controls (NCs). The expression of miR-106a, thrombospondin 2 (THBS2), Bax, cleaved caspase-3 and Bcl-2, cell viability, relative caspase-3 activity and TNF-α, IL-1ß, IL-6 content were respectively detected by quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, Cell Counting Kit-8 (CCK-8) and enzyme linked immunosorbent assay (ELISA). The relationship between miR-106a and THBS2 was confirmed by dual luciferase reporter assay. RESULTS: MiR-106a was up-regulated in serum of sepsis patients, CLP-induced mice models and LPS-induced TCMK-1 cells. LPS reduced cell viability and Bcl-2 expression, and increased caspase-3 activity, Bax expression, the content of TNF-α, IL-1ß, IL-6. THBS2 was a target of miR-106a. The decreases of caspase-3 activity, TNF-α, IL-1ß, IL-6, Bax expression and the increases of cell viability, Bcl-2 expression caused by miR-106a knockdown were reversed when THBS2 silencing in LPS-stimulated TCMK-1 cells. CONCLUSION: MiR-106a aggravated LPS-induced inflammation and apoptosis of TCMK-1 cells via regulating THBS2 expression.

R-spondin-3 induces secretory, antimicrobial Lgr5+ cells in the stomach.

Wnt signalling stimulated by binding of R-spondin (Rspo) to Lgr-family members is crucial for gastrointestinal stem cell renewal. Infection of the stomach with Helicobacter pylori stimulates increased secretion of Rspo by myofibroblasts, leading to an increase in proliferation of Wnt-responsive Axin2+Lgr5- stem cells in the isthmus of the gastric gland and finally gastric gland hyperplasia. Basal Lgr5+ cells are also exposed to Rspo3, but their response remains unclear. Here, we demonstrate that-in contrast to its known mitogenic activity-Rspo3 induces differentiation of basal Lgr5+ cells into secretory cells that express and secrete antimicrobial factors, such as intelectin-1, into the lumen. The depletion of Lgr5+ cells or the knockout of Rspo3 in myofibroblasts leads to hypercolonization of the gastric glands with H. pylori, including the stem cell compartment. By contrast, systemic administration or overexpression of Rspo3 in the stroma clears H. pylori from the gastric glands. Thus, the Rspo3-Lgr5 axis simultaneously regulates both antimicrobial defence and mucosal regeneration.

Thrombospondin enhances RANKL-dependent osteoclastogenesis and facilitates lung cancer bone metastasis.

Lung cancers have a predilection for metastasizing to bone. The matricellular glycoprotein thrombospondin (TSP)-2 regulates multiple biological functions and has a critical role in tumor development and metastasis, although its effects are uncertain in lung cancer bone metastasis. This study demonstrates that TSP-2 expression is highly correlated with lung cancer tumor stage and that the TSP-2 neutralizing antibody reduces osteoclast formation in conditioned medium obtained from lung cancer cells. We also found that TSP-2 promotes osteoclastogenesis through the RANKL-dependent pathway and that TSP-2-mediated osteoclastogenesis involves the transactivation of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) via the inhibition of miR-486-3p expression. Osteoblasts played a critical role in osteoclast differentiation and incubation of osteoblasts with TSP-2 altered the RANKL:OPG ratio. Furthermore, TSP-2 knockdown inhibited lung cancer osteolytic metastasis in vivo. TSP-2 appears to be worth targeting for the prevention of bone metastasis in lung cancer.

Regulation of steroidogenic function of luteal cells by thrombospondin and insulin in water buffalo (Bubalus bubalis).

The present study examined the effect of exogenous thrombospondin 1 (TSP1) on the steroidogenic function of luteal cells cultured invitro. Furthermore, the transcriptional interaction of insulin with TSP1 and its receptor, cluster of differentiation 36 (CD36) were also investigated. At the highest dose (500ngmL-1) TSP1 significantly downregulated the expression of the angiogenic marker von Willebrand factor (vWF) and progesterone production in cultured luteal cells. Moreover, the simultaneous upregulation in the expression of caspase 3 by exogenous TSP1 was consistent with a reduction in the number of viable luteal cells as determined by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltertrazolium bromide (MTT) assay after 72h of culture. However, the expression of critical enzymes in the progesterone synthetic pathway was not significantly modulated by treatment with TSP1 in cultured luteal cells. Knocking out of endogenous TSP1 with the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPRassociated protein9 (Cas9) system improved the viability of luteal cells as well as increasing progesterone production and decreasing caspase 3 activation. Insulin treatment suppressed the expression of TSP1 and CD36 in cultured luteal cells in a dose- and time-dependent manner. To conclude, TSP1 acts as a negative endogenous regulator of angiogenesis that attenuates progesterone production, possibly by reducing the number of luteal cells via apoptosis during luteal regression, whereas insulin as a luteinising signal may have inhibited the thrombospondin system for the efficient development of luteal function.

MiR-106a aggravates sepsis-induced acute kidney injury by targeting THBS2 in mice model

Abstract Purpose To investigate the role and related mechanisms of miR-106a in sepsis-induced AKI. Methods Serum from sepsis and healthy patients was collected, sepsis mouse model was established by cecal ligation and puncture (CLP). TCMK-1 cells were treated with lipopolysaccharide (LPS) and transfected with THBS2-small interfering RNA (siTHBS2), miR-106a inhibitor, miR-106a mimics and their negative controls (NCs). The expression of miR-106a, thrombospondin 2 (THBS2), Bax, cleaved caspase-3 and Bcl-2, cell viability, relative caspase-3 activity and TNF-α, IL-1β, IL-6 content were respectively detected by quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, Cell Counting Kit-8 (CCK-8) and enzyme linked immunosorbent assay (ELISA). The relationship between miR-106a and THBS2 was confirmed by dual luciferase reporter assay. Results MiR-106a was up-regulated in serum of sepsis patients, CLP-induced mice models and LPS-induced TCMK-1 cells. LPS reduced cell viability and Bcl-2 expression, and increased caspase-3 activity, Bax expression, the content of TNF-α, IL-1β, IL-6. THBS2 was a target of miR-106a. The decreases of caspase-3 activity, TNF-α, IL-1β, IL-6, Bax expression and the increases of cell viability, Bcl-2 expression caused by miR-106a knockdown were reversed when THBS2 silencing in LPS-stimulated TCMK-1 cells. Conclusion MiR-106a aggravated LPS-induced inflammation and apoptosis of TCMK-1 cells via regulating THBS2 expression.

Efficient Generation of Small Intestinal Epithelial-like Cells from Human iPSCs for Drug Absorption and Metabolism Studies.

The small intestine plays an important role in the absorption and metabolism of oral drugs. In the current evaluation system, it is difficult to predict the precise absorption and metabolism of oral drugs. In this study, we generated small intestinal epithelial-like cells from human induced pluripotent stem cells (hiPS-SIECs), which could be applied to drug absorption and metabolism studies. The small intestinal epithelial-like cells were efficiently generated from human induced pluripotent stem cell by treatment with WNT3A, R-spondin 3, Noggin, EGF, IGF-1, SB202190, and dexamethasone. The gene expression levels of small intestinal epithelial cell (SIEC) markers were similar between the hiPS-SIECs and human adult small intestine. Importantly, the gene expression levels of colonic epithelial cell markers in the hiPS-SIECs were much lower than those in human adult colon. The hiPS-SIECs generated by our protocol exerted various SIEC functions. In conclusion, the hiPS-SIECs can be utilized for evaluation of drug absorption and metabolism.

R-spondin3-LGR4 signaling protects hepatocytes against DMOG-induced hypoxia/reoxygenation injury through activating ß-catenin.

BACKGROUND & AIMS: Leucine-rich repeat G-protein-coupled receptor 4 (LGR4) and its ligands R-spondin1-4 (Rspos) have been vastly investigated in embryonic development. The biological functions of Rspos-LGR4 system in liver remains largely unknown. Here, we explored whether it protects hepatocytes against hypoxia/reoxygenation (H/R) induced damage. METHODS: H/R injury was induced by dimethyloxalylglycine (DMOG) in AML12 cells and the effects of Rspo3 on cell proliferation and apoptosis were assessed. Specific shRNAs were used to interfere LGR4 or ß-catenin. RESULTS: DMOG caused hepatocytes damage evidenced by increase in HIF-1α, cell death and apoptosis genes p27 and Bax, with concurrent decrease of cell proliferation genes PCNA and CyclinD1. Of all the Rspos, Rspo3 is predominantly expressed in AML12 hepatocytes. Importantly, Rspo3 demonstrated an alteration in a manner similar to proliferation-related genes during H/R injury. Rspo3 pretreatment rendered hepatocytes less vulnerable to DMOG induced H/R injury. Ablation of LGR4 using shRNA attenuated the protective effects of Rspo3. Wnt3a also protected AML12 cells from damages caused by H/R, showing enhanced proliferation activity. Notably, knockdown of ß-catenin in hepatocytes completely abolished the effect of Rspo3 pretreatment on the expression levels of PCNA and CyclinD1. CONCLUSION: Rspo3-LGR4 axis protects hepatocytes from H/R injury via activating ß-catenin.

Methylphenidate enhances neuronal differentiation and reduces proliferation concomitant to activation of Wnt signal transduction pathways.

Methylphenidate (Ritalin) is the most commonly prescribed drug in the treatment of attention-deficit hyperactivity disorder. It is suggested that in vivo, methylphenidate treatment supports cortical maturation, however, the molecular and cellular mechanisms are not well understood. This study aimed to explore the potential effect of methylphenidate on cell proliferation and maturation in various cellular models, hypothesizing its interaction with the Wnt-signaling. The termination of cell proliferation concomitant to neuronal maturation following methylphenidate treatment was observed in all of the cell-models tested: murine neural stem-, rat PC12- and the human SH-SY5Y-cells. Inhibition of Wnt-signaling in SH-SY5Y cells with Dkk1 30 min before methylphenidate treatment suppressed neuronal differentiation but enhanced proliferation. The possible involvement of the dopamine-transporter in cell differentiation was discounted following the observation of opposing results after GBR-12909 treatment. Moreover, Wnt-activation via methylphenidate was confirmed in Wnt-luciferase-reporter assay. These findings reveal a new mechanism of action of methylphenidate that might explain long-term effects.

Therapeutic Role for TSP-2 Antibody in a Murine Asthma Model.

BACKGROUND: Specific immunotherapy, including agonists for Toll-like receptor 2 (TLR2), have been shown to protect from allergies and to have a high immunomodulatory capacity. METHODS: A new antibody, TSP-2, reactive against an epitope of the extracellular domain of TLR2, was identified. The effect of the antibody on dendritic cells was assessed by immunohistochemistry, Western blot, and flow cytometric analysis. The effect of TSP-2 in a murine asthma model induced with ovalbumin (OVA) was assessed. The model is a form of airway hyperresponsiveness (AHR) and was analyzed by whole-body plethysmography, the measurement of Th1/Th2 cytokines in bronchial alveolar lavage fluid (BALF) and serum by ELISA, and the CCK-8 assay for lymphocyte proliferation. The effect of TSP-2 on the maturation of bone marrow-derived dendritic cells (BMDCs) was assessed by flow cytometric analysis. RESULTS: TSP-2 promoted the maturation of dendritic cells and the proliferation of lymphocyte in vitro and in vivo. The effect of TSP-2 on T helper 1 (Th1)/Th2 cytokine secretion was slightly more powerful than that of Pam3CSK4. TSP-2 antibody reduced AHR and OVA-specific IgE levels in allergic asthma. TSP-2 antibody also reduced lung inflammation and decreased leukocyte numbers in an OVA-sensitized and challenged asthma model. TSP-2 antibody increased OVA-stimulated I-A, CD80, CD86, and MHC-II levels on BMDCs. CONCLUSIONS: This study identifies a novel therapeutic strategy for AHR, which uses antibodies reactive against TLR2. It also provides theoretical evidence for the control of allergic asthma by targeting TLR2.

R-Spondin1 expands Paneth cells and prevents dysbiosis induced by graft-versus-host disease.

The intestinal microbial ecosystem is actively regulated by Paneth cell-derived antimicrobial peptides such as α-defensins. Various disorders, including graft-versus-host disease (GVHD), disrupt Paneth cell functions, resulting in unfavorably altered intestinal microbiota (dysbiosis), which further accelerates the underlying diseases. Current strategies to restore the gut ecosystem are bacteriotherapy such as fecal microbiota transplantation and probiotics, and no physiological approach has been developed so far. In this study, we demonstrate a novel approach to restore gut microbial ecology by Wnt agonist R-Spondin1 (R-Spo1) or recombinant α-defensin in mice. R-Spo1 stimulates intestinal stem cells to differentiate to Paneth cells and enhances luminal secretion of α-defensins. Administration of R-Spo1 or recombinant α-defensin prevents GVHD-mediated dysbiosis, thus representing a novel and physiological approach at modifying the gut ecosystem to restore intestinal homeostasis and host-microbiota cross talk toward therapeutic benefits.

Thrombospondin-4 divergently regulates voltage-gated Ca2+ channel subtypes in sensory neurons after nerve injury.

Loss of high-voltage-activated (HVA) calcium current (ICa) and gain of low-voltage-activated (LVA) ICa after painful peripheral nerve injury cause elevated excitability in sensory neurons. Nerve injury is also accompanied by increased expression of the extracellular matrix glycoprotein thrombospondin-4 (TSP4), and interruption of TSP4 function can reverse or prevent behavioral hypersensitivity after injury. We therefore investigated TSP4 regulation of ICa in dorsal root ganglion (DRG) neurons. During depolarization adequate to activate HVA ICa, TSP4 decreases both N- and L-type ICa and the associated intracellular calcium transient. In contrast, TSP4 increases ICa and the intracellular calcium signal after low-voltage depolarization, which we confirmed is due to ICa through T-type channels. These effects are blocked by gabapentin, which ameliorates neuropathic pain by targeting the α2δ1 calcium subunit. Injury-induced changes of HVA and LVA ICa are attenuated in TSP4 knockout mice. In the neuropathic pain model of spinal nerve ligation, TSP4 application did not further regulate ICa of injured DRG neurons. Taken together, these findings suggest that elevated TSP4 after peripheral nerve injury may contribute to hypersensitivity of peripheral sensory systems by decreasing HVA and increasing LVA in DRG neurons by targeting the α2δ1 calcium subunit. Controlling TSP4 overexpression in peripheral sensory neurons may be a target for analgesic drug development for neuropathic pain.

Exogenous R-Spondin1 Induces Precocious Telogen-to-Anagen Transition in Mouse Hair Follicles.

R-spondin proteins are novel Wnt/ß-catenin agonists, which signal through their receptors leucine-rich repeat-containing G-protein coupled receptor (LGR) 4/5/6 and substantially enhance Wnt/ß-catenin activity. R-spondins are reported to function in embryonic development. They also play important roles in stem cell functions in adult tissues, such as the intestine and mammary glands, which largely rely on Wnt/ß-catenin signaling. However, in the skin epithelium and hair follicles, the information about R-spondins is deficient, although the expressions and functions of their receptors, LGR4/5/6, have already been studied in detail. In the present study, highly-enriched expression of the R-spondin family genes (Rspo1/2/3/4) in the hair follicle dermal papilla is revealed. Expression of Rspo1 in the dermal papilla is specifically and prominently upregulated before anagen entry, and exogenous recombinant R-spondin1 protein injection in mid-telogen leads to precocious anagen entry. Moreover, R-spondin1 activates Wnt/ß-catenin signaling in cultured bulge stem cells in vitro, changing their fate determination without altering the cell proliferation. Our pioneering study uncovers a role of R-spondin1 in the activation of cultured hair follicle stem cells and the regulation of hair cycle progression, shedding new light on the governance of Wnt/ß-catenin signaling in skin biology and providing helpful clues for future treatment of hair follicle disorders.