Gene expression profiling of molecular target genes which are regulated by hepatocyte nuclear factor 1 Alpha (HNF1A) in rat insulin-secreting cells: role in HNF1A-MODY

In this project, we were interested in studying HNF1A-induced defects in the expression of genes involved in the differentiation and regeneration of beta-cells in a cellular model of HNFlA-maturity-onset-diabetes-of-the-young(HNF1A-MODY), as well as the signaling pathways leading to such effects. Inactivating mutations in the human gene encoding transcription factor-1 (tcf-l), also known as hepatocyte nuclear factor 1 (hnf) 1a, result in decreased beta-cell mass, defects in glucose-induced insulin secretion, and HNF1A-MODY, the most common monogenic form of diabetes. We utilised rat INS-1 cells to conditionally overexpress wild-type HNF1A, the frameshift Pro291fsinsC-HNF1A mutant (a common human HNF1A-MODY mutation), and DN-HNFlA all of which were under the control of a reverse tetracycline-dependent transactivator system. Additionally, we utilised paraffin-embedded pancreatic sections on slides from the Rat Insulin Promoter (RIP)-DN-HNF1A transgenic mice and pancreatic sections of the healthy islets of wild-type C57BL/6JBomTac mice as an in-vivo model. Genes involved in beta-cell differentiation and regeneration were investigated by high density oligonucleotide Affymetrix microarrays the Pro29lfsinsC-HNFlA induced INS-1 cells. These GeneChip expression arrays enabled us to simultaneously monitor genome-wide expression profiles and identify known and novel HNFlA dependent target genes which are involved in a variety of physiological processes. These putative genes were confirmed using qPCR and Western blotting. We identified a prominent down-regulation of Bone Morphogenic Protein (bmp-3) mRNA expression in induced vs. non-induced mutant INS-1 cells. In contrast, the inducible expression of WT-HNF1A led to a time-dependent increase in bmp-3 mRNA levels. QPCR and Western blotting analysis also identified a compensatory upregulation of bmp-r2 gene/protein and alk4 gene expression in Pro29lfsinsC-HNF1A cells, and coincided with a time-dependent reduction in the expression of the insulin. BMPs represent a large family of growth factors critically involved in tissue differentiation and development, which signal via type I and type I1 BMP receptors. Treatment of INS-1 cells with recombinant BMP-3 restored insulin expression levels which were decreased by the HNF1A frame-shift mutation. This coincided with an increase in the phosphorylation of SMAD 2/3. Furthermore, immunohistochemistry on DN-HNF1A transgenic mice revealed a reduction in double positive staining of BMP-3 / Insulin, in insulin positive cells. Finally, we could demonstrate decreased activation of the bmp-3 promoter using a luciferase construct containing the bmp-3 promoter following expression of the ProfsinsC-HNF1A mutant. These findings suggest a critical link between HNF1A-MODY-induced alterations in gene expression and growth factors that control tissue differentiation and regeneration. We also identified a significant up-regulation of Pancreatitis Associated Protein III (PAP III) mRNA expression in induced INS-1 cells. We found that of all the reg genes examined (PAP I, PAP II PAP III, and PSP/reg), the expression of Pro29lfsinsCHNF1A mutant led to a rapid and potent induction PSP/reg. Interestingly, PAP III and PSP/reg belong to the \u22Reg\u22 (Regenerating) family of genes and have been implicated in islet regeneration. We detected a prominent induction of PSP/reg at gene and protein level during DN-HNF1A-induced apoptosis of INS-1 cells, and this induction was inhibited by the caspase inhibitor, zVAD.fmk. Conditioned culture medium from DN-HNF1A-expressing cells, but not from DN-HNF1A-expressing cells treated with zVAD.fmk, was sufficient to induce PSP/reg gene expression in naïve, INS-1 cells. Filtration and flow cytometry experiments suggested that Annexin-V-positive microparticles originating from blebbing membranes of apoptosing INS-1 cells mediated this effect. Treatment with recombinant PSP/reg protein stimulated cell proliferation and reversed the DN-HNF1A-induced decrease in p27 and insulin gene expression. We propose that apoptotic beta-cells secrete microparticles that stimulate PSP/reg induction in neighbouring cells, and facilitate the recovery of beta-cell mass. Collectively, these findings provide key insights into a primary aspect of beta-cell mass regulation, and could open up new avenues for renewal of impaired or decreased beta-cell mass in HNF1A-MODY and Type 2 diabetes

Emerging Roles of Glycogen Synthase Kinase 3 in the Treatment of Brain Tumors

The constitutively active protein glycogen synthase kinase 3 (GSK3), a serine/threonine kinase, acts paradoxically as a tumor suppressor in some cancers while potentiates growth in others. Deciphering what governs its actions is vital for understanding many pathological conditions, including brain cancer. What are seemingly disparate roles of GSK3 stems from the complex regulation of many cellular functions by GSK3. This review focuses on the regulation of GSK3, its role in survival, apoptosis and DNA damage, and finally its potential therapeutic impact in brain cancer. A thorough understanding of this versatile protein is critical for improving the outcome of various diseases, especially cancer

Effects of hepatocyte nuclear factor-1A and -4A on pancreatic stone protein/regenerating protein and C-reactive protein gene expression: implications for maturity-onset diabetes of the young

BACKGROUND: There is a significant clinical overlap between patients with hepatocyte nuclear factor (HNF)-1A and HNF4A maturity-onset diabetes of the young (MODY), two forms of monogenic diabetes. HNF1A and HNF4A are transcription factors that control common and partly overlapping sets of target genes. We have previously shown that elevated serum pancreatic stone protein / regenerating protein A (PSP/reg1A) levels can be detected in subjects with HNF1A-MODY. In this study, we investigated whether PSP/reg is differentially regulated by HNF1A and HNF4A. METHODS: Quantitative real-time PCR (qPCR) and Western blotting were used to validate gene and protein expression in cellular models of HNF1A- and HNF4A-MODY. Serum PSP/reg1A levels and high-sensitivity C-reactive protein (hsCRP) were measured by ELISA in 31 HNF1A- and 9 HNF4A-MODY subjects. The two groups were matched for age, body mass index, diabetes duration, blood pressure, lipid profile and aspirin and statin use. RESULTS: Inducible repression of HNF1A and HNF4A function in INS-1 cells suggested that PSP/reg induction required HNF4A, but not HNF1A. In contrast, crp gene expression was significantly reduced by repression of HNF1A, but not HNF4A function. PSP/reg levels were significantly lower in HNF4A subjects when compared to HNF1A subjects [9.25 (7.85-12.85) ng/ml vs. 12.5 (10.61-17.87) ng/ml, U-test P = 0.025]. hsCRP levels were significantly lower in HNF1A-MODY [0.22 (0.17-0.35) mg/L] compared to HNF4A-MODY group [0.81 (0.38-1.41) mg/L, U-test P = 0.002], Parallel measurements of serum PSP/reg1A and hsCRP levels were able to discriminate HNF1A- and HNF4A-MODY subjects. CONCLUSION: Our study demonstrates that two distinct target genes, PSP/reg and crp, are differentially regulated by HNF1A and HNF4A, and provides clinical proof-of-concept that serum PSP/reg1A and hsCRP levels may distinguish HNF1A-MODY from HNF4A-MODY subjects

Serum levels of pancreatic stone protein (PSP)/reg1A as an indicator of beta-cell apoptosis suggest an increased apoptosis rate in hepatocyte nuclear factor 1 alpha (HNF1A-MODY) carriers from the third decade of life onward

Background Mutations in the transcription factor hepatocyte nuclear factor-1-alpha (HNF1A) result in the commonest type of maturity onset diabetes of the young (MODY). HNF1A-MODY carriers have reduced pancreatic beta cell mass, partially due to an increased rate of apoptosis. To date, it has not been possible to determine when apoptosis is occurring in HNF1A-MODY.We have recently demonstrated that beta cell apoptosis stimulates the expression of the pancreatic stone protein/regenerating (PSP/reg) gene in surviving neighbour cells, and that PSP/reg1A protein is subsequently secreted from these cells. The objective of this study was to determine whether serum levels of PSP/reg1A are elevated during disease progression in HNF1A-MODY carriers, and whether it may provide information regarding the onset of beta-cell apoptosis. Methods We analysed serum PSP/reg1A levels and correlated with clinical and biochemical parameters in subjects with HNF1A-MODY, glucokinase (GCK-MODY), and type 1 diabetes mellitus. A control group of normoglycaemic subjects was also analysed. Results PSP/reg1A serum levels were significantly elevated in HNF1A-MODY (n = 37) subjects compared to controls (n = 60) (median = 12.50 ng/ml, IQR = 10.61-17.87 ng/ml versus median = 10.72 ng/ml, IQR = 8.94-12.54 ng/ml, p = 0.0008). PSP/reg1A correlated negatively with insulin levels during OGTT, (rho = −0.40, p = 0.02). Interestingly we noted a significant positive correlation of PSP/reg1A with age of the HNF1A-MODY carriers (rho = 0.40 p = 0.02) with an age of 25 years separating carriers with low and high PSP/reg1A levels. Patients with type 1 diabetes mellitus also had elevated serum levels of PSP/reg1A compared to controls, however this was independent of the duration of diabetes. Conclusion Our data suggest that beta cell apoptosis contributes increasingly to the pathophysiology of HNF1A-MODY in patients 25 years and over. PSP/reg1A may be developed as a serum marker to detect increased beta-cell apoptosis, or its therapeutic response

AMP kinase–mediated activation of the BH3-only protein Bim couples energy depletion to stress-induced apoptosis

Disturbances in cellular ion gradients by excitotoxicity promote apoptosis through activation of the Bcl-2 family member Bim

Adhesion Class GPCRs (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

Adhesion GPCRs are structurally identified on the basis of a large extracellular region, similar to the Class B GPCR, but which is linked to the 7TM region by a GPCR autoproteolysis-inducing (GAIN) domain [8] containing a GPCR proteolytic site. The N-terminus often shares structural homology with adhesive domains (e.g. cadherins, immunolobulin, lectins) facilitating inter- and matricellular interactions and leading to the term adhesion GPCR [82, 332]. Several receptors have been suggested to function as mechanosensors [254, 234, 315, 32]. The nomenclature of these receptors was revised in 2015 as recommended by NC-IUPHAR and the Adhesion GPCR Consortium [100]

Adhesion Class GPCRs in GtoPdb v.2023.1

Adhesion GPCRs are structurally identified on the basis of a large extracellular region, similar to the Class B GPCR, but which is linked to the 7TM region by a GPCR autoproteolysis-inducing (GAIN) domain [10] containing a GPCR proteolysis site (GPS). The N-terminal extracellular region often shares structural homology with adhesive domains (e.g. cadherins, immunolobulin, lectins) facilitating inter- and matricellular interactions and leading to the term adhesion GPCR [104, 418]. Several receptors have been suggested to function as mechanosensors [320, 288, 396, 38]. Cryo-EM structures of the 7-transmembrane domain of several adhesion GPCRs have been determined recently [292, 21, 403, 212, 300, 302, 431, 293]. The nomenclature of these receptors was revised in 2015 as recommended by NC-IUPHAR and the Adhesion GPCR Consortium [125]