Jatuporn Sujjitjoon

Defective PAX4 R192H transcriptional repressor activities associated with maturity onset diabetes of the young and early onset-age of type 2 diabetes

AIMS PAX4 R192H polymorphism was reported to be associated with maturity onset diabetes of the young (MODY) and early onset-age of type 2 diabetes (T2D). This study aimed to evaluate transcriptional repression activity of PAX4 R192H polymorphism on its target promoters comparing with wild-type PAX4. METHODS Wild-type PAX4 and PAX4 R192H proteins were expressed in vitro and the cell compartmentalization of each protein was examined after transfection of the plasmid constructs into βTC3 cells followed by Western-blot analysis. The plasmid containing wild-type PAX4 or PAX4 R192H was co-transfected into βTC3 and αTC-1.9 cells with insulin or glucagon promoter-reporter construct. Transcriptional repression activities were then determined by dual-luciferase reporter assay. RESULTS Wild-type PAX4 and PAX4 R192H, which were found to be equally expressed in vitro and transfection systems, were present in the nuclear compartment. Transcriptional repressor activities of PAX4 R192H on human insulin and glucagon promoters were reduced when they were compared with those of wild-type PAX4. CONCLUSIONS These results suggested that PAX4 R192H polymorphism generated a protein with defect in transcriptional repressor activities on its target genes, which may lead to β-cell dysfunction associated with MODY and early onset-age of T2D as reported in our previous study.

Aberrant mRNA splicing of paired box 4 ( PAX4 ) IVS7-1G>A mutation causing maturity-onset diabetes of the young, type 9

AimsPaired box 4 (PAX4) mutations cause maturity-onset diabetes of the young, type 9 (MODY9). The molecular defect and alteration of PAX4 function associated with the mutation PAX4 IVS7-1G>A in a family with MODY9 and severe diabetic complications were studied.MethodsWe investigated the functional consequences of PAX4 IVS7-1G>A on mRNA splicing using minigene assays. Wild-type and mutant PAX4 were expressed in mouse pancreatic β- and α-cell lines, and protein levels and translocation of PAX4 into the nucleus were determined. We also examined transcriptional repression of PAX4 target-gene promoters and β-cell viability under diabetic-like (high-glucose) conditions.ResultsPAX4 IVS7-1G>A disrupts an acceptor splice site, causing an adjacent cryptic splice site within exon 8 to be used, resulting in a three-nucleotide deletion and glutamine deletion at position 250 (p.Q250del). Wild-type and PAX4 Q250del proteins were expressed at similar levels and could translocate normally into the nucleus in βTC3 and αTC1.9 cells. However, the repressor functions of PAX4 Q250del on human insulin and glucagon promoters in INS-1 832/13 and αTC1.9 cells were significantly decreased, compared with that of wild-type PAX4. Moreover, the rate of apoptosis was increased in INS-1 cells over-expressing PAX4 Q250del when cultured in high-glucose conditions.ConclusionsPAX4 IVS7-1G>A caused aberrant mRNA splicing and PAX4 Q250 deletion. The mutation impaired PAX4 repressor functions on target-gene promoters and increased susceptibility to apoptosis upon high glucose exposure. Thus, PAX4 IVS7-1G>A contributes to the pathogenesis of diabetes in this MODY9 family through β-cell dysfunction.

Novel Adiponectin Variants Identified in Type 2 Diabetic Patients Reveal Multimerization and Secretion Defects

ADIPOQ, encoding adiponectin, is a candidate gene for type 2 diabetes (T2D) identified by genome-wide linkage analyses with supporting evidence showing the protein function in sensitizing insulin actions. In an endeavor to characterize candidate genes causing T2D in Thai patients, we identified 10 novel ADIPOQ variations, several of which were non-synonymous variations observed only in the patients. To examine the impact of these non-synonymous variations on adiponectin structure and biochemical characteristics, we conducted a structural analysis of the wild-type and variant proteins by in silico modeling and further characterized biochemical properties of the variants with predicted structural abnormalities from the modeling by molecular and biochemical studies. The recombinant plasmids containing wild-type and variant ADIPOQ cDNAs derived from the variations identified by our study (R55H, R112H, and R131H) and previous work (G90S and R112C) were constructed and transiently expressed and co-expressed in cultured HEK293T cells to investigate their oligomerization, interaction, and secretion. We found that the novel R55H variant impaired protein multimerization but it did not exert the effect over the co-expressed wild-type protein while novel R131H variant impaired protein secretion and also affected the co-expressed wild-type protein in a dominant negative fashion. The R131H variant could traffic from the endoplasmic reticulum to the Golgi, trans-Golgi network, and early endosome but could not be secreted. The R131H variant was likely to be degraded through the lysosomal system and inhibition of its degradation rescued the variant protein from secretion defect. We have shown the possibility of using in silico modeling for predicting the effect of amino acid substitution on adiponectin oligomerization. This is also the first report that demonstrates a dominant negative effect of the R131H variant on protein secretion and the possibility of using protein degradation inhibitors as therapeutic agents in the patients carrying adiponectin variants with secretion defect.

PAX4 R192H and P321H polymorphisms in type 2 diabetes and their functional defects

We have previously identified PAX4 mutations causing MODY9 and a recent genome-wide association study reported a susceptibility locus of type 2 diabetes (T2D) near PAX4. In this study, we aim to investigate the association between PAX4 polymorphisms and T2D in Thai patients and examine functions of PAX4 variant proteins. PAX4 rs2233580 (R192H) and rs712701 (P321H) were genotyped in 746 patients with T2D and 562 healthy normal control subjects by PCR and restriction-fragment length polymorphism method. PAX4 variant proteins were investigated for repressor function on human insulin and glucagon promoters and for cell viability and apoptosis upon high glucose exposure. Genotype and allele frequencies of PAX4 rs2233580 were more frequent in patients with T2D than in control subjects (P=0.001 and 0.0006, respectively) with odds ratio of 1.66 (P=0.001; 95% confidence interval, 1.22–2.27). PAX4 rs712701 was not associated with T2D but it was in linkage disequilibrium with rs2233580. The 192H/321H (A/A) haplotype was more frequent in T2D patients than in controls (9.5% vs 6.6%; P=0.009). PAX4 R192H, but not PAX4 P321H, impaired repression activities on insulin and glucagon promoters and decreased transcript levels of genes required to maintain β-cell function, proliferation and survival. Viability of β-cell was reduced under glucotoxic stress condition for the cells overexpressing either PAX4 R192H or PAX4 P321H or both. Thus these PAX4 polymorphisms may increase T2D risk by defective transcription regulation of target genes and/or decreased β-cell survival in high glucose condition.

Functional defect of truncated hepatocyte nuclear factor-1α (G554fsX556) associated with maturity-onset diabetes of the young

A novel frameshift mutation attributable to 14-nucleotide insertion in hepatocyte nuclear factor-1alpha (HNF-1alpha) encoding a truncated HNF-1alpha (G554fsX556) with 76-amino acid deletion at its carboxyl terminus was identified in a Thai family with maturity-onset diabetes of the young (MODY). The wild-type and mutant HNF-1alpha proteins were expressed by in vitro transcription and translation (TNT) assay and by transfection in HeLa cells. The wild-type and mutant HNF-1alpha could similarly bind to human glucose-transporter 2 (GLUT2) promoter examined by electrophoretic mobility shift assay (EMSA). However, the transactivation activities of mutant HNF-1alpha on human GLUT2 and rat L-type pyruvate kinase (L-PK) promoters in HeLa cells determined by luciferase reporter assay were reduced to approximately 55-60% of the wild-type protein. These results suggested that the functional defect of novel truncated HNF-1alpha (G554fsX556) on the transactivation of its target-gene promoters would account for the beta-cell dysfunction associated with the pathogenesis of MODY.

Estradiol Prevents High Glucose-Induced β-cell Apoptosis by Decreased BTG2 Expression

Hyperglycemia stimulates several pathways to induce pancreatic β-cell apoptosis. In our previous study by mRNA analysis, we demonstrated that B-cell translocation gene 2 (BTG2) expression was up-regulated in INS-1 cells cultured under high glucose conditions, but this effect was reversed by estrogen. In the present study, we demonstrated that BTG2 mRNA and protein expressions in both INS-1 cells and mouse pancreatic islets increased under high glucose conditions compared to those cultured under basal glucose conditions, while in the presence of estrogen, the BTG2 mRNA and protein expressions decreased. SiRNA-BTG2 significantly reduced cell apoptosis, cleaved-caspase 3, and Bax, compared to the siRNA-control in INS-1 cultured under high glucose conditions. We further demonstrated that BTG2 promoter activity was activated under high glucose conditions whereas estrogen significantly reduced it. The effects of estrogen on BTG2 expression were inhibited by estrogen receptor inhibitors. Also, under high glucose conditions, p53 and Bax mRNA and protein expressions increased, but they decreased in the presence of estrogen. Again, the effect of estrogen on p53 and Bax expression was inhibited by estrogen receptor inhibitors. Taken together, this study demonstrates that estrogen reduces pancreatic β-cell apoptosis under high glucose conditions via suppression of BTG2, p53, and Bax expressions.

Cytotoxic activity of effector T cells against cholangiocarcinoma is enhanced by self-differentiated monocyte-derived dendritic cells

Cholangiocarcinoma (CCA) is a cancer of the bile ducts that is associated with poor prognosis and poor treatment outcome. Approximately one-third of CCA patients can undergo surgery, but the recurrence rate is high and chemotherapy often cannot satisfactorily prolong survival. Cellular immunotherapy based on adoptive T-cell transfer is a potential treatment for CCA; however, the development of this technology and the search for an appropriate tumor-associated antigen are still ongoing. To enhance the cytotoxic activity of effector T cells against CCA, we developed self-differentiated monocyte-derived dendritic cells (SD-DC) presenting cAMP-dependent protein kinase type I-alpha regulatory subunit (PRKAR1A), which is an overexpressed protein that plays a role in the regulation of tumor growth to activate T cells for CCA cell killing. Dendritic cells (DCs) transduced with lentivirus harboring tri-cistronic cDNA sequences (SD-DC-PR) could produce granulocyte–macrophage colony-stimulating factor, interleukin-4, and PRKAR1A. SD-DC showed similar phenotypes to those of DCs derived by conventional method. Autologous effector T cells (CD3+, CD8+) activated by SD-DC-PR exhibited greater cytotoxic activity against CCA than those activated by conventionally-derived DCs. Effector T cells activated by SD-DC-PR killed 60% of CCA cells at an effector-to-target ratio of 15:1, which is approximately twofold greater than the cell killing performance of those stimulated with control DC. The cytotoxic activities of effector T cells activated by SD-DC-PR against CCA cells were significantly associated with the expression levels of PRKR1A in CCA cells. This finding that SD-DC-PR effectively stimulated autologous effector T cells to kill CCA cells may help to accelerate the development of novel therapies for treating CCA.

Abstract 3150: GD2-specific chimeric antigen receptor T cells targeting retinoblastoma

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Introduction: Retinoblastoma (RB) is an aggressive childhood cancer of high incidence (1/20,000). The conventional treatment is enucleation followed by chemotherapy. Patients with poor prognosis often face resistant relapses after chemo- and radiation therapies. Immunotherapy based on chimeric antigen receptor (CAR)-engineered T cells targeting cancer-specific antigens has shown great potential in treating hematological malignancies. This study aims to identify RB-specific surface antigens and investigate the RB tumor targeting function of a 4th generation CAR-T cells with specificity for disialoganglioside 2 (GD2) protein. Methods: We examined RB surgical specimens from eight patients for GD2 expression by immunohistochemistry, and a RB cell line (Y79RB) by flow cytometry. Activated T-cells were engineered to express a 4th generation CAR specific to GD2 protein by using lentiviral transduction. Y79RB cells were labeled with a green fluorescence protein and used as target cells in a CAR-T/RB co-culture experiment. Tumor cell lysis was monitored under a fluorescent microscope, and apoptotic cell death was analyzed by AnnexinV/PI staining and flow cytometry. Results: All eight RB tumor specimens were strongly positive for GD2 based on immunohistochemistry. In addition, GD2 expression was detected on the cell surface of Y79RB at a very high level by flow cytometry analysis as compared with known GD2 -positive tumor cells such as neuroblastoma and Ewings sarcoma. In vitro co-culture experiments showed that Y79RB cells were targeted and killed by GD2 CAR-Ts but not control CD19 CAR-Ts. At a ratio of 1:1, the RB cells were completely eradicated after 3 days of co-culture with GD2 CAR-Ts. When we increased the tumor cell ratio in the coculture, we observed tumor resistance to GD2 CAR-Ts. Further analysis of the escaped Y79RB cells indicated that their surface GD2 expression was down-regulated. In addition, we found that PD-L1 was up-regulated in Y79RB cells when cocultured with GD2 CAR-Ts but not control CD19 CAR-Ts. We further demonstrated that the increased PD-L1 expression in Y79RB interacted with PD-1 on activated CAR-Ts and suppressed the CAR-T effector functions. Conclusion: This is the first report that GD2-specific CAR-Ts can effective target and kill RB. Decreased CAR-T killing efficiency may occur due to the up-regulation of PD-L1 and down-regulation of GD2 expressions on the tumor cells. The combination of GD2-specific CAR-Ts and anti-PD-L1 antibody may improve the CAR-T immunotherapy efficacy. Citation Format: Jatuporn Sujjitjoon, La-ong Sri Atchaneeyasakul, Shih-Ting Tsao, Elias Sayour, Pa-thai Yenchitsomanus, Lung-Ji Chang. GD2-specific chimeric antigen receptor T cells targeting retinoblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3150. doi:10.1158/1538-7445.AM2015-3150