T-cell Death-associated Gene Research Articles

GDF10 is a negative regulator of vascular calcification

Cardiovascular mortality is particularly high and increasing in patients with chronic kidney disease, with vascular calcification (VC) as a major pathophysiologic feature. VC is a highly regulated biological process similar to bone formation involving osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs). We have previously demonstrated that loss of T-cell death-associated gene 51 (TDAG51) expression leads to an attenuation of medial VC. We now show a significant induction of circulating levels of growth differentiation factor 10 (GDF10) in TDAG51-/- mice, which was of interest due to its established role as an inhibitor of osteoblast differentiation. The objective of this study was to examine the role of GDF10 in the osteogenic transdifferentiation of VSMCs. Using primary mouse and human VSMCs, as well as exvivo aortic ring cultures, we demonstrated that treatment with recombinant human (rh) GDF10 mitigated phosphate-mediated hydroxyapatite (HA) mineral deposition. Furthermore, exvivo aortic rings from GDF10-/- mice exhibited increased HA deposition compared to C57BL/6J controls. To explain our observations, we identified that rhGDF10 treatment reduced protein expression of runt-related transcription factor 2, a key driver of osteogenic transdifferentiation of VSMCs and VC. In support of these findings, invivo treatment with rhGDF10 attenuated VD3-induced VC. Furthermore, we demonstrated an increase in circulating GDF10 in patients with chronic kidney disease with clinically defined severe VC, as assessed by coronary artery calcium score. Thus, our studies identify GDF10 as a novel inhibitor of mineral deposition and as such, may represent a potential novel biomarker and therapeutic target for the detection and management of VC.

Heat Shock Related Protein Expression in Abdominal Testes of Asian Elephant (Elephas maximus).

The abdominal testes of Asian elephants show normal spermatogenesis. Heat shock in cryptorchid testes elevates heat shock factor (HSF) expression, leading to germ cell apoptosis, while increased heat shock proteins (HSPs) levels provide protection. To investigate how heat shock affects elephant spermatogenic cells, focusing on heat shock-related molecules and the cell death mechanism, immunohistochemistry and TUNEL staining were employed to assess the immunoexpression of several heat shock-related molecules and the status of apoptosis in elephant fibroblasts (EF) induced by heat shock stimulus. Additionally, the immunoexpression of heat shock-related molecules and cell proliferation status in the elephant spermatogenic cells. Our finding indicated that heat shock-induced HSF1 immunoexpression in EF leads to apoptosis mediated by T-cell death-associated gene 51 (TDAG51) while also upregulating HSP70 to protect damaged cells. In elephant spermatogenic cells, immunostaining revealed a predominance of proliferating cell nuclear antigen (PCNA)-positive cells with minimal TDAG51- and TUNEL-positive cells, suggesting active proliferation and apoptosis suppression during normal spermatogenesis in the abdominal testis. Interestingly, spermatogonia co-immunoexpressed HSF1 and HSP90, potentially reducing apoptosis through protective mechanisms different from those observed in other mammals. Spermatogenic cells did not show immunolocalisation of HSP70, and hence, it may not contribute to protecting the spermatogonia from heat shock because the transcriptional activity of HSF1 is suppressed by HSP90A binding. This study provides insight into the specific heat shock response and defence mechanisms in elephant spermatogenic cells and may contribute to our understanding of species-specific adaptation to environmental stresses of the testis.

Targeting astrocytic TDAG8 with delayed CO2 postconditioning improves functional outcomes after controlled cortical impact injury in mice

T-cell death-associated gene 8 (TDAG8), a G-protein-coupled receptor sensing physiological or weak acids, regulates inflammatory responses. However, its role in traumatic brain injury (TBI) remains unknown. Our recent study showed that delayed CO2 postconditioning (DCPC) has neuroreparative effects after TBI. We hypothesized that activating astrocytic TDAG8 is a key mechanism for DCPC. WT and TDAG8−/− mice received DCPC daily by transiently inhaling 10% CO2 after controlled cortical impact (CCI). HBAAV2/9-GFAP-m-TDAG8-3xflag-EGFP was used to overexpress TDAG8 in astrocytes. The beam walking test, mNSS, immunofluorescence and Golgi-Cox staining were used to evaluate motor function, glial activation and dendritic plasticity. DCPC significantly improved motor function; increased total dendritic length, neuronal complexity and spine density; inhibited overactivation of astrocytes and microglia; and promoted the expression of astrocytic brain-derived neurotrophic factor in WT but not TDAG8−/− mice. Overexpressing TDAG8 in astrocytes surrounding the lesion in TDAG8−/− mice restored the beneficial effects of DCPC. Although the effects of DCPC on Days 14–28 were much weaker than those of DCPC on Days 3–28 in WT mice, these effects were further enhanced by overexpressing astrocytic TDAG8. Astrocytic TDAG8 is a key target of DCPC for TBI rehabilitation. Its overexpression is a strategy that broadens the therapeutic window and enhances the effects of DCPC.

TDAG51 promotes transcription factor FoxO1 activity during LPS-induced inflammatory responses.

Tight regulation of Toll-like receptor (TLR)-mediated inflammatory responses is important for innate immunity. Here, we show that T-cell death-associated gene 51 (TDAG51/PHLDA1) is a novel regulator of the transcription factor FoxO1, regulating inflammatory mediator production in the lipopolysaccharide (LPS)-induced inflammatory response. TDAG51 induction by LPS stimulation was mediated by the TLR2/4 signaling pathway in bone marrow-derived macrophages (BMMs). LPS-induced inflammatory mediator production was significantly decreased in TDAG51-deficient BMMs. In TDAG51-deficient mice, LPS- or pathogenic Escherichia coli infection-induced lethal shock was reduced by decreasing serum proinflammatory cytokine levels. The recruitment of 14-3-3ζ to FoxO1 was competitively inhibited by the TDAG51-FoxO1 interaction, leading to blockade of FoxO1 cytoplasmic translocation and thereby strengthening FoxO1 nuclear accumulation. TDAG51/FoxO1 double-deficient BMMs showed significantly reduced inflammatory mediator production compared with TDAG51- or FoxO1-deficient BMMs. TDAG51/FoxO1 double deficiency protected mice against LPS- or pathogenic E. coli infection-induced lethal shock by weakening the systemic inflammatory response. Thus, these results indicate that TDAG51 acts as a regulator of the transcription factor FoxO1, leading to strengthened FoxO1 activity in the LPS-induced inflammatory response.

TDAG51 Attenuates Impaired Lipid Metabolism and Insulin Resistance in Gestational Diabetes Mellitus Through SREBP-1/ANGPTL8 Pathway.

T-cell death-associated gene 51 (TDAG51) belongs to the transcription factor family and is involved in the energy homeostasis of the liver through the regulation of lipogenesis. To evaluate the role of T-cell death-associated gene 51 in gestational diabetes mellitus. Experimental study. A total of 30 patients with GDM were enrolled in the study. TDAG51 expression in patients with gestational diabetes mellitus was assessed by Western blotting and quantitative reverse-transcription polymerase chain reaction. A high-fat and high-sugar diet was used to establish a Gestational diabetes mellitus model. gestational diabetes mellitus mice were injected with lentivirus-mediated overexpression of TDAG51. Blood glucose was measured using a glucometer, and glucose and insulin tolerance tests were performed to detect insulin resistance. Liver and adipose tissues were subjected to hematoxylineosin staining. Cell apoptosis was detected by TUNEL staining. Human villous trophoblast cells (HTR-8/SVneo) were treated with a high-glucose medium to induce gestational diabetes mellitus. TDAG51 was downregulated in gestational diabetes mellitus and high glucose-induced HTR-8/SVneo. TDAG51 overexpression reduced the level of blood glucose, enhanced serum insulin, and attenuated glucose and insulin tolerance in gestational diabetes mellitus mice. TDAG51 overexpression also ameliorated impaired lipid metabolism and alleviated adipocyte hypertrophy and hepatic lipid droplets in gestational diabetes mellitus mice. The expressions of SREBP-1 and ANGPTL8 were upregulated in gestational diabetes mellitus and showed a negative correlation with TDAG51 in patients with gestational diabetes mellitus. TDAG51 increased the expressions of SREBP-1 and ANGPTL8 in gestational diabetes mellitus mice. TDAG51 overexpression reduced cell apoptosis and enhanced cell viability of high glucose-induced HTR-8/SVneo. Ectopic expression of ANGPTL8 attenuated the TDAG51-induced increase in cell viability and decrease in apoptosis in high glucose-induced HTR-8/SVneo. TDAG51 alleviated impaired lipid metabolism and insulin resistance in gestational diabetes mellitus via downregulation of SREBP 1/ANGPTL8 pathway.

TDAG51 deficiency attenuates dextran sulfate sodium-induced colitis in mice

Inflammatory bowel disease (IBD), including ulcerative colitis and Crohn’s disease, is a group of chronic inflammatory diseases of the gastrointestinal tract. Although the multifactorial etiology of IBD pathogenesis is relatively well documented, the regulatory factors that confer a risk of IBD pathogenesis remain less explored. In this study, we report that T-cell death-associated gene 51 (TDAG51/PHLDA1) is a novel regulator of the development of dextran sulfate sodium (DSS)-induced colitis in mice. TDAG51 expression was elevated in the colon tissues of DSS-induced experimental colitis mice. TDAG51 deficiency protected mice against acute DSS-induced lethality and body weight changes and disease severity. DSS-induced structural damage and mucus secretion in colon tissues were significantly reduced in TDAG51-deficient mice compared with wild-type mice. We observed similar results in a DSS-induced chronic colitis mouse model. Finally, we showed that the production of inflammatory mediators, including proinflammatory enzymes, molecules and cytokines, was decreased in DSS-treated TDAG51-deficient mice compared with DSS-treated wild-type mice. Thus, we demonstrated that TDAG51 deficiency plays a protective role against DSS-induced colitis by decreasing the production of inflammatory mediators in mice. These findings suggest that TDAG51 is a novel regulator of the development of DSS-induced colitis and is a potential therapeutic target for IBD.

Research Progress of Pleckstrin Homology Like Domain Family A Member 1 in Tumor

Pleckstrin homology like domain family A member 1(PHLDA1) is also known as T-cell death-associated gene 51 (TDAG51).Studies have demonstrated that the abnormal expression of PHLDA1 is closely associated with the formation,development,and metastasis of tumors.We summarized the latest research advances in the structure and biological properties of PHLDA1,as well as the roles of PHLDA1 in multiple malignanttumors such as breast cancer,cancer,liver gastric cancer,liver cancer,melanoma,and osteosarcoma,aiming to comprehensively reveal the significance of PHLDA1 in the clinical diagnosis of tumors.

Ethylenediaminetetraacetic acid (EDTA) enhances cAMP production in human TDAG8-expressing cells

Ethylenediaminetetraacetic acid (EDTA) is a chelating agent that binds tightly to metal ions. We found that cAMP response element (CRE)-driven promoter activity by protons was enhanced by EDTA in human T-cell death-associated gene 8 (TDAG8)-overexpressed HEK293T cells. The enhancing action by EDTA was also detected by proton-induced cAMP production that is located upstream from the CRE-driven promoter activity even at physiological proton concentration pH7.4. The proton-induced CRE-driven promoter activity was not enhanced by other chelating agents, ethylene glycol tetraacetic acid (EGTA) and sodium citrate. The enhanced CRE-driven promoter activity by EDTA was not attenuated by increasing the extracellular calcium ion concentration. These results indicate that the EDTA-enhancing action may not be due to its chelating action but might rather be another EDTA-specific effect. Enhanced cAMP production by EDTA was also detected in a human leukemia cell line HL-60, in which TDAG8 and OGR1 (ovarian cancer G-protein-coupled receptor 1) were endogenously expressed, suggesting that the medical use of EDTA would influence the physiological and pathophysiological functions of hematopoietic cells.

Gut Mucosal Microbiome Is Perturbed in Rheumatoid Arthritis Mice and Partly Restored after TDAG8 Deficiency or Suppression by Salicylanilide Derivative.

Rheumatoid arthritis (RA), an autoimmune disease, is characterized by chronic joint inflammation and pain. We previously found that the deletion of T-cell death-associated gene 8 (TDAG8) significantly reduces disease severity and pain in RA mice. Whether it is by modulating gut microbiota remains unclear. In this study, 64 intestinal samples of feces, cecal content, and cecal mucus from the complete Freund’s adjuvant-induced arthritis mouse models were compared. The α- and β-diversity indices of the microbiome were significantly lower in RA mice. Cecal mucus showed a higher ratio of Firmicutes to Bacteroidetes in RA than healthy mice, suggesting the ratio could serve as an RA indicator. Four core genera, Eubacterium_Ventriosum, Alloprevotella, Rikenella, and Treponema, were reduced in content in both feces and mucus RA samples, and could serve microbial markers representing RA progression. TDAG8 deficiency decreased the abundance of proinflammation-related Eubacterium_Xylanophilum, Clostridia, Ruminococcus, Paraprevotella, and Rikenellaceae, which reduced local mucosal inflammation to relieve RA disease severity and pain. The pharmacological block of the TDAG8 function by a salicylanilide derivative partly restored the RA microbiome to a healthy composition. These findings provide a further understanding of specific bacteria interactions with host gut mucus in the RA model. The modulation by TDAG8 on particular bacteria can facilitate microbiota-based therapy.

TDAG51-Deficiency Podocytes are Protected from High-Glucose-Induced Damage Through Nrf2 Activation via the AKT-GSK-3β Pathway.

T cell death-associated gene 51 (TDAG51) has been implicated in the development of various pathological conditions. However, whether TDAG51 plays a role in diabetic renal disease remains unknown. The current work investigated the possible function of TDAG51 in diabetic renal disease using high-glucose (HG)-stimulated podocytes in vitro. The elevation of TDAG51 was observed in podocytes in response to HG exposure and the glomeruli of diabetic mice. The siRNAs targeting TDAG51 were applied to deplete TDAG51 in HG-stimulated podocytes. Crucially, TDAG51 deficiency was sufficient to decrease the apoptosis, oxidative stress, and inflammation caused by HG. Mechanically, the inhibition of TDAG51 was capable of enhancing the activation of nuclear factor E2-related factor 2 (Nrf2) associated with the upregulation of AKT-glycogen synthase kinase-3β (GSK-3β) pathway. The reduction of AKT abolished the activation of Nrf2 elicited by TDAG51 deficiency. Additionally, the reduction of Nrf2 diminished the anti-HG injury effect elicited by TDAG51 deficiency. Overall, these data demonstrate that TDAG51 deficiency defends against HG-induced podocyte damage through Nrf2 activation by regulating AKT-GSK-3β pathway. This study suggests that TDAG1 may have a potential role in diabetic renal disease by affecting HG-induced podocyte damage.

Extracellular acidity in tumor tissue upregulates programmed cell death protein1 expression on tumor cells via proton-sensing G protein-coupled receptors.

Acidity in the tumor microenvironment has been reported to promote cancer growth and metastasis. In our study, we examined a potential relation between extracellular acidity and expression level of the immune checkpoint molecule programmed cell death protein1 (PD-L1) in murine squamous cell carcinoma (SCC) and melanoma cell lines. PD-L1 expression in the tumor cells was upregulated by culturing in a low pH culture medium. Tumor-bearing mice were allowed to ingest sodium bicarbonate, resulting in neutralization of acidity in the tumor tissue, a decrease in PD-L1 expression in tumor cells and suppression of tumor growth in vivo. Proton-sensing G protein-coupled receptors, T-cell death-associated gene 8 (TDAG8) and ovarian cancer G-protein-coupled receptor 1 (OGR1), were upregulated by low pH, and essentially involved in the acidity-induced elevation of PD-L1 expression in the tumor cells. Human head and neck SCC RNAseq data from the Cancer Genome Atlas also suggested a statistically significant correlation between expression levels of the proton sensors and PD-L1 mRNA expression. These findings strongly suggest that neutralization of acidity in tumor tissue may result in reduction of PD-L1 expression, potentially leading to inhibition of an immune checkpoint and augmentation of antitumor immunity.

T-Cell Death Associated Gene 51 Is a Novel Negative Regulator of PPARγ That Inhibits PPARγ-RXRα Heterodimer Formation in Adipogenesis

The nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) is the master transcriptional regulator in adipogenesis. PPARγ forms a heterodimer with another nuclear receptor, retinoid X receptor (RXR), to form an active transcriptional complex, and their transcriptional activity is tightly regulated by the association with either coactivators or corepressors. In this study, we identified T-cell death-associated gene 51 (TDAG51) as a novel corepressor of PPARγ-mediated transcriptional regulation. We showed that TDAG51 expression is abundantly maintained in the early stage of adipogenic differentiation. Forced expression of TDAG51 inhibited adipocyte differentiation in 3T3-L1 cells. We found that TDAG51 physically interacts with PPARγ in a ligand-independent manner. In deletion mutant analyses, large portions of the TDAG51 domains, including the pleckstrin homology-like, glutamine repeat and proline-glutamine repeat domains but not the proline-histidine repeat domain, are involved in the interaction with the region between residues 140 and 506, including the DNA binding domain, hinge, ligand binding domain and activation function-2 domain, in PPARγ. The heterodimer formation of PPARγ-RXRα was competitively inhibited in a ligand-independent manner by TDAG51 binding to PPARγ. Thus, our data suggest that TDAG51, which could determine adipogenic cell fate, acts as a novel negative regulator of PPARγ by blocking RXRα recruitment to the PPARγ-RXRα heterodimer complex in adipogenesis.

The protective role of proton-sensing TDAG8 in the brain injury in a mouse ischemia reperfusion model

Extracellular acidification in the brain has been observed in ischemia; however, the physiological and pathophysiological implications of the pH reduction remain largely unknown. Here, we analyzed the roles of proton-sensing G protein-coupled receptors, including T-cell death-associated gene 8 (TDAG8), ovarian cancer G protein-coupled receptor 1 (OGR1), and G protein-coupled receptor 4 (GPR4) in a mouse ischemia reperfusion model. Cerebral infarction and dysfunctional behavior with transient middle cerebral artery occlusion (tMCAO) and subsequent reperfusion were exacerbated by the deficiency of TDAG8, whereas no significant effect was observed with the deficiency of OGR1 or GPR4. We confirmed that the pH of the predicted infarction region was 6.5. TDAG8 mRNA was observed in Iba1-positive microglia in the mouse brain. The tMCAO increased the mRNA expression of tumor necrosis factor-α in the ipsilateral cerebral hemisphere and evoked morphological changes in microglia in an evolving cerebral injury. These tMCAO-induced actions were significantly enhanced by the TDAG8 deficiency. Administration of minocycline, which is known to inhibit microglial activation, improved the cerebral infarction and dysfunctional behavior induced by tMCAO in the TDAG8-deficient mouse. Thus, acidic pH/TDAG8 protects against cerebral infarction caused by tMCAO, at least due to the mechanism involving the inhibition of microglial functions.

TDAG51 (T-Cell Death-Associated Gene 51) Is a Key Modulator of Vascular Calcification and Osteogenic Transdifferentiation of Arterial Smooth Muscle Cells.

Cardiovascular disease is the primary cause of mortality in patients with chronic kidney disease. Vascular calcification (VC) in the medial layer of the vessel wall is a unique and prominent feature in patients with advanced chronic kidney disease and is now recognized as an important predictor and independent risk factor for cardiovascular and all-cause mortality in these patients. VC in chronic kidney disease is triggered by the transformation of vascular smooth muscle cells (VSMCs) into osteoblasts as a consequence of elevated circulating inorganic phosphate (Pi) levels, due to poor kidney function. The objective of our study was to investigate the role of TDAG51 (T-cell death-associated gene 51) in the development of medial VC. Using primary mouse and human VSMCs, we found that TDAG51 is induced in VSMCs by Pi and is expressed in the medial layer of calcified human vessels. Furthermore, the transcriptional activity of RUNX2 (Runt-related transcription factor 2), a well-established driver of Pi-mediated VC, is reduced in TDAG51-/- VSMCs. To explain these observations, we identified that TDAG51-/- VSMCs express reduced levels of the type III sodium-dependent Pi transporter, Pit-1, a solute transporter, a solute transporter, a solute transporter responsible for cellular Pi uptake. Significantly, in response to hyperphosphatemia induced by vitamin D3, medial VC was attenuated in TDAG51-/- mice. Our studies highlight TDAG51 as an important mediator of Pi-induced VC in VSMCs through the downregulation of Pit-1. As such, TDAG51 may represent a therapeutic target for the prevention of VC and cardiovascular disease in patients with chronic kidney disease.

Temporal expression patterns of distinct cytokines and M1/M2 macrophage polarization regulate rheumatoid arthritis progression.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of synovial joints and often associated with chronic pain. Chronic joint inflammation is attributed to severe proliferation of synoviocytes and resident macrophages and infiltration of immune cells. These cells secrete pro-inflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin 6 (IL-6) and IL-17 to overcome actions of anti-inflammatory cytokines, thereby maintaining chronic inflammation and pain. The imbalance between pro-inflammatory cytokines (produced by M1 macrophages) and anti-inflammatory cytokines (produced by M2 macrophages) is a feature of RA progression, but the switch time of M1/M2 polarization and which receptor regulates the switch remain unsolved. Here we used an established RA mouse model to demonstrate that TNF-α expression was responsible for the initial acute stage of inflammation and pain (1-4weeks), IL-17 expression the transition stage (4-12weeks), and IL-6 expression the later maintenance stage (> 12weeks). The switch time of M1/M2 polarization occurred at 4-8weeks. We also identified a potential compound, anthra[2,1-c][1,2,5] thiadiazole-6,11-dione (NSC745885), that specifically inhibited T-cell death-associated gene 8 (TDAG8) function and expression. NSC745885 decreased joint inflammation and destruction and attenuated pain by reducing cytokine production and regulating the M1/M2 polarization switch. TDAG8 may participate in regulating the M1/M2 polarization and temporal expression of distinct cytokines to control RA progression.

T-cell death-associated gene 8 accelerates atherosclerosis by promoting vascular smooth muscle cell proliferation and migration

Background and aimsAtherosclerosis is a serious cardiovascular disease, featuring inflammation, abnormal proliferation and migration of vascular smooth muscle cells (VSMCs). During atherosclerosis, inflammation may cause low pH. T-cell death-associated gene 8 (Tdag8) is a proton-sensing receptor, however, the role of Tdag8 in VSMCs remains unknown. This study aimed to investigate the potential effects of Tdag8 in VSMCs during atherosclerosis. MethodsWe examined the expression of Tdag8 in an atherosclerotic model of high-fat-diet-fed ApoE−/− mice, while the role and mechanism of Tdag8 in phenotype transformation, proliferation and migration of VSMCs were investigated in a series of in vivo and in vitro experiments. ResultsWe first found that Tdag8 expression at the mRNA and protein level was significantly increased in atherosclerotic ApoE−/− mice. Immunofluorescence staining showed that Tdag8 was primarily distributed in PCNA-positive VSMCs and the phenotype of VSMCs switching from contractile phenotype to synthetic phenotype. Additionally, the protein level of Tdag8 was upregulated in FBS-treated VSMCs. VSMCs proliferation and migration were inhibited by Tdag8 silencing and increased by Tdag8 overexpression. Further mechanistic studies showed that cAMP level was increased in Tdag8-overexpressing VSMCs and ApoE−/− mice. However, the PKA inhibitor H-89 reversed Tdag8-induced VSMC proliferation and migration. ConclusionsThe results demonstrate that Tdag8 mediated phenotype transformation, proliferation and migration of VSMCs via the cAMP/PKA signaling pathway, thus partially contributing to atherosclerosis.

Expression of proton-sensing G-protein-coupled receptors in selected skin tumors.

In humans, there are four known proton-sensing G-Protein-coupled receptors (pH-GPCRs): GPR4 (GPR19), TDAG8 (GPR65, T-cell death-associated gene 8), OGR1 (GPR68, ovarian cancer GPCR1) and G2A (GPR132, G2 accumulation protein). They are known to be involved in sensing changes of extracellular proton concentrations in the acidic microenvironment of tumors, which leads to altered cell proliferation, migration, metastasis, immune cell function and inflammation. However, little is known about the expression of pH-GPCRs in the skin and especially skin cancers. We studied the expression of pH-GPCRs in selected skin cancers, that is Merkel cell carcinoma (MCC), dermatofibrosarcoma protuberans (DFSP), atypical fibroxanthoma (AFX) and pleomorphic dermal sarcoma (PDS). We did immunohistochemistry and immunofluorescence to analyse the expression of GPR4, TDAG8, OGR1 and G2A using paraffin-embedded tissue samples (n=4, exceptions: PDS GPR4/GPR65 n=5, AFX GPR132 n=3) from patients suffering from MCC, DFSP, AFX and PDS. (a) GPR4 was expressed on all AFX and PDS specimens. All AFX and MCC showed a positive expression of G2A. All PDS exhibited a strong positive expression of G2A. (b) MCCs neither expressed GPR4 nor TDAG8. All DFSP showed no expression of TDAG8. (c) For any other combination of GPCR and skin disease, we found positive/negative mixed results. These are the first results on pH-GPCRs in selected skin cancers. We provide evidence that these GPCRs are differentially expressed on the various types of skin cancers and that they can potentially be addressed as a therapeutic target in extensive disease.

CD10, TDAG51, CK20, AR, INSM1, and Nestin Expression in the Differential Diagnosis of Trichoblastoma and Basal Cell Carcinoma.

Trichoblastoma (TB) and basal cell carcinoma (BCC) are 2 different neoplasms composed of basaloid cells and have overlapping histopathological features. We compared the immunoexpression of CD10, T-cell death-associated gene 51 (TDAG51), cytokeratin 20 (CK20), androgen receptor (AR), insulinoma-associated protein 1 (INSM1), and nestin for the differential diagnosis of these tumors. We assessed a total of 27 BCC and 27 TB cases, including 4 TB lesions in nevus sebaceous and 3 malignant TB lesions for CD10, TDAG51, CK20, AR, INSM1, and nestin expression. Staining for CK20, TDAG51, INSM1, and stromal CD10 was significantly more common in TB cases than in BCC cases ( P < .001). Epithelial CD10 and AR staining was significantly more common in BCC cases than in TB cases ( P < .001). The difference between the groups for nestin staining was not significant ( P > .05). Stromal CD10 staining was the most sensitive marker (96.3%) and INSM1 the least sensitive (55.6%) marker for TB. TDAG51 showed 100% specificity for TB. A larger number of CK20 positive cells was found in the cases associated with nevus sebaceous than in the other TBs. All the selected markers except nestin were useful for the differential diagnosis between TB and BCC. CD10 and TDAG51 were more useful than the other markers. The use of CK20 could be preferred in nevus sebaceous lesions. INSM1 was less effective in highlighting Merkel cells within the lesion than CK20.

TDAG51 is a key modulator of vascular calcification and osteogenic transdifferentiation of arterial smooth muscle cells

Cardiovascular disease (CVD) is the primary cause of morbidity and mortality in patients with chronic kidney disease (CKD). More than half of all deaths from patients with end-stage renal disease (ESRD) requiring dialysis can be attributed to CVD. Although traditional risk factors for CVD are prevalent in ESRD, they cannot fully explain the high mortality rates. Vascular calcification (VC) in the medial layer of the vessel wall is a unique and prominent feature in patients with advanced CKD and is now recognized as an important predictor and an independent risk factor for cardiovascular and all-cause and mortality in these patients. VC in CKD is triggered by vascular smooth muscle cells (VSMCs) transforming into osteoblasts, a consequence of elevated circulating phosphate levels due to poor kidney function. Understanding and targeting this SMC trans-differentiation and subsequent VC represents a viable therapeutic avenue for managing CKD complications. Similar to bone formation, VC is a highly regulated process that draws many parallels with osteogenic differentiation. We have recently observed that T-cell Death Associated Gene 51 (TDAG51), a protein upregulated by oxidative and endoplasmic reticulum stress, is induced in SMCs under conditions of hyperphosphatemia and is expressed in the medial layer of calcified human vessels. We have found that ablation of TDAG51 reduces SMC trans-differentiation and VC, both in human SMCs treated with TDAG51 siRNA and in mouse VSMCs isolated from TDAG51−/− aortas. To explain these findings, we observed that TDAG−/− VSMCs express reduced levels of the of the Pit-1, a solute transporter responsible for cellular phosphate uptake. Consistent with this observation, reduced Pit-1 expression was associated with reduced intracellular phosphate levels and reduced phosphate-induced apoptosis. Further, TDAG−/− VSMCs exhibit significantly reduced transcriptional activity of a phosphate-inducible transcription factor, and well-established driver of VC, known as Runx2. Support or Funding Information This work was supported in part by research grants to Richard C. Austin from the Heart and Stroke Foundation of Ontario (T-6146), the Heart and Stroke Foundation of Canada (G-13-0003064 and G-15-0009389) and the Canadian Institutes of Health Research (74477). TDAG51 is involved in the development and progression of VC through the regulation of intracellular phosphate content and Runx2 transcriptional activity. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Restoration of hepatic TDAG51 expression improves insulin signaling and reduces weight gain in mouse models of non‐alcoholic fatty liver disease

The absence of T‐cell death‐associated gene 51 (TDAG51) in mice results in late‐onset insulin resistance (IR), obesity and hepatic steatosis. In support of these findings, mice fed a high fat diet, which leads to IR and obesity, as well as the leptin‐deficient mouse model, a well‐established genetic model of type 2 diabetes and obesity, show significantly diminished hepatic TDAG51 protein levels. Here we report that hepatic TDAG51 protein levels are reduced in multiple mouse models of liver injury and in liver biopsies from patients with non‐alcoholic steatohepatitis. Given that hepatic TDAG51 protein levels decrease under conditions of fatty liver and correlate with IR, we investigated whether restoring hepatic TDAG51 protein levels would reverse hepatic IR. Hepatic expression of a TDAG51‐GFP fusion protein was increased via a liver‐specific adeno‐associated virus (AAV) in both TDAG51 knockout and leptin‐deficient mice. Restoration of hepatic TDAG51 protein increased insulin sensitivity and reduced weight gain compared to mice injected with a control AAV vector encoding only GFP. Specifically, TDAG51 knockout mice expressing ectopic TDAG51 demonstrated: 1) improved Akt473 phosphorylation, 2) reduced expression of diacylglycerol O‐acyltransferase, which catalyzes the final step in triglyceride biosynthesis, and 3) reduced expression of the SREBP‐1c target genes, fatty acid synthase and stearoyl‐CoA desaturase‐1. These findings demonstrate that restoring hepatic TDAG51 in mouse models of metabolic disease and fatty liver improves hepatic insulin signaling and reduces weight gain. However, this protective effect is lost over time, correlates with fatty liver progression and is accompanied by diminished hepatic TDAG51 protein stability. Taken together, these studies suggest that hepatic TDAG51 is an important factor that mediates insulin signaling and weight gain. Future studies aimed at increasing hepatic TDAG51 protein levels and/or stability may represent a new therapeutic opportunity for the amelioration of obesity and augmentation of insulin sensitivity in non‐alcoholic fatty liver disease.Support or Funding InformationR.C.A is supported by grants from the Heart and Stroke Foundation of Ontario and the Canadian Institutes of Health Research. Financial support from St. Joseph's Healthcare Hamilton is also acknowledged. R.C.A. is a Career Investigator of the Heart and Stroke Foundation of Ontario and holds the Amgen Canada Research Chair in Nephrology. K.N.M acknowledges financial support from the William R. Hummel Homocystinuria Research Fund and holds the Ehst‐Hummel‐Kaufmann Family Endowed Chair in Inherited Metabolic Disease. G.R.S is supported by a Canada Research Chair in Metabolism and Obesity, the J. Bruce Duncan Endowed Chair in Metabolic Diseases and grants from the Canadian Institutes of Health Research and Diabetes Canada.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.