Michael S. Lan

IA1 is NGN3-dependent and essential for differentiation of the endocrine pancreas

Neurogenin 3 (Ngn3) is key for endocrine cell specification in the embryonic pancreas and induction of a neuroendocrine cell differentiation program by misexpression in adult pancreatic duct cells. We identify the gene encoding IA1, a zinc‐finger transcription factor, as a direct target of Ngn3 and show that it forms a novel branch in the Ngn3‐dependent endocrinogenic transcription factor network. During embryonic development of the pancreas, IA1 and Ngn3 exhibit nearly identical spatio‐temporal expression patterns. However, embryos lacking Ngn3 fail to express IA1 in the pancreas. Upon ectopic expression in adult pancreatic duct cells Ngn3 binds to chromatin in the IA1 promoter region and activates transcription. Consistent with this direct effect, IA1 expression is normal in embryos mutant for NeuroD1, Arx, Pax4 and Pax6, regulators operating downstream of Ngn3. IA1 is an effector of Ngn3 function as inhibition of IA1 expression in embryonic pancreas decreases the formation of insulin‐ and glucagon‐positive cells by 40%, while its ectopic expression amplifies neuroendocrine cell differentiation by Ngn3 in adult duct cells. IA1 is therefore a novel Ngn3‐regulated factor required for normal differentiation of pancreatic endocrine cells.

Value of Antibodies to Islet Protein Tyrosine Phosphatase–Like Molecule in Predicting Type 1 Diabetes

Islet antigens associated with type 1 diabetes include a recently identified protein tyrosine phosphatase–like molecule IA-2, which contains the intracellular fragment IA-2ic. To determine whether combinations of antibodies including those to IA-2 characterize and predict type 1 diabetes, we studied antibodies to IA-2, IA-2ic, glutamic acid decarboxylase (GAD65), and islet cell antibodies (ICAs) in 1) 60 newly diagnosed type 1 diabetic patients followed for 1 year, 2) 31 monozygotic twin pairs discordant for type 1 diabetes followed up to 12 years (11 twins developed diabetes), 3) 18 dizygotic twin pairs discordant for type 1 diabetes, and 4) normal healthy control subjects. Newly diagnosed type 1 diabetic patients frequently had antibodies to IA-2 (62%), IA-2ic (67%), GAD65 (77%), and ICAs (85%). The intracellular fragment of IA-2 probably contains the immunodominant epitope as 137 of 143 samples with IA-2 antibodies from type 1 diabetic patients also had IA-2ic antibodies. Monozygotic twins were usually discordant for antibody specificities. Concordance was higher in monozygotic than matched dizygotic twins for both antibody combinations (33 vs. 6%, P < 0.05) and the development of diabetes (33 vs. 0%, P < 0.01). In monozygotic twins, all the antibodies were highly predictive of type 1 diabetes (positive predictive values all >87%), although antibodies were also detected in twins at low risk of disease. In summary, IA-2 emerges as a major antigen associated with type 1 diabetes and distinct from GAD65. Type 1 diabetes–associated autoimmunity, which is probably induced by environmental factors, does not necessarily herald progression to the disease. However, genetic factors may influence the development of combinations of disease-associated antibodies and the progression to type 1 diabetes.

NeuroD1/E47 regulates the E-box element of a novel zinc finger transcription factor, IA-1, in developing nervous system

IA-1 is a novel zinc finger transcription factor with a restricted tissue distribution in the embryonic nervous system and tumors of neuroendocrine origin. The 1.7-kilobase 5′-upstream DNA sequence of the human IA-1 gene directed transgene expression predominantly in the developing nervous system including forebrain, midbrain, hindbrain, spinal cord, retina, olfactory bulb, and cerebellum, which recapitulated the expression patterns of neuroendocrine tissues and childhood brain tumors. The IA-1 promoter deletion reporter gene constructs revealed that the sequence between -426 and -65 bp containing three putative E-boxes (∼361 bp) upstream of the transcription start site was sufficient to confer tissue-specific transcriptional activity. Further mutation analysis revealed that the proximal E-box (E3) closest to the start site is critical to confer transcriptional activity. Electrophoretic mobility shift assay and transient transfection studies demonstrated that the NeuroD1 and E47 heterodimer are the key transcription factors that regulate the proximal E-box of the IA-1 promoter. Therefore, we concluded that the IA-1 gene is developmentally expressed in the nervous system and the NeuroD1/E47 transcription factors up-regulate IA-1 gene expression through the proximal E-box element of the IA-1 promoter.

Autoantibodies to IA-2 in IDDM: Location of Major Antigenic Determinants

Thirty-three IDDM sera that immunoprecipitated fulllength IA-2 were tested for reactivity with different fragments of the IA-2 molecule. The fragments were prepared by PCR amplification of IA-2 cDNA and by expression in a rabbit reticulocyte transcription/translation system. Whereas all 33 sera reacted with the intracellular domain (amino acid 604 to 979), none of the sera reacted with the extracellular domain of IA-2 (amino acid 31 to 577). Analysis of the reactivity of IDDM sera with the different regions of the intracellular domain showed that 94% (31 of the 33) reacted with the COOH-terminus (amino acid 771 to 979), 40% reacted with the NH2-terminus (amino acid 604 to 776), and 40% reacted with the middle portion (amino acid 692 to 875). Of the 31 sera that reacted with the COOH-terminus, 14 of these reacted only with the COOH-terminus and with no other region. Of the 13 sera that reacted with the NH2-terminus, only one reacted exclusively with the NH2-terminus. Treatment of the different domains of IA-2 with trypsin showed that only the COOH-terminus was resistant to trypsin, arguing that it is from this region of the IA-2 molecule that the 40-kDa tryptic fragment from insulinoma cells is derived. From these experiments, it is concluded that the major antigenic determinant of IA-2 is located at the COOH-terminus and that minor antigenic determinants are located at the NH2-terminus and middle portion of the intracellular domain.

Structure, expression, and biological function of INSM1 transcription factor in neuroendocrine differentiation

Zinc‐finger transcription factors are DNAbinding proteins that are implicated in many diverse biological functions. INSM1 (formerly IA‐1) contains five zinc‐finger motifs and functions as a transcription factor. INSM1 protein structure is highly conserved in homologues of different species. It is predominantly expressed in developing neuroendocrine tissues and the nervous system in mammals. INSM1 represents an important player in early embryonic neurogenesis. In pancreatic endocrine cell differentiation, Ngn3 first activates INSM1 and subsequently NeuroD/P2. Conversely, INSM1 exerts a feedback mechanism to suppress NeuroD/P2 and its own gene expression. INSM1 gene ablation in the mouse results in the impairment of pancreatic endocrine cell maturation. Further, deletion of INSM1 severely impairs catecholamine biosynthesis and secretion from the adrenal gland that results in early embryonic lethality. Genetically, INSM1 acts as a downstream factor of Mash 1 and Phox2b in the differentiation of the sympatho‐adrenal lineage. In the developing neocortex, mouse embryos lacking INSM1 expression contain half the number of basal progenitors and show a reduction in cortical plate radial thickness. Cell signaling studies reveal that INSM1 contributes to the induction of cell cycle arrest/exit necessary to facilitate cellular differentiation. INSM1 is highly expressed in tumors of neuroendocrine origin. Hence, its promoter could serve as a tumor‐specific promoter that drives a specific targeted cancer gene therapy for the treatment of neuroendocrine tumors. Taken together, all of these features of INSM1 strongly support its role as an important regulator during neuroendocrine differentiation.—Lan, M. S., Breslin, M. B. Structure, expression, and biological function of INSM1 transcription factor in neuroendocrine differentiation. FASEB J. 23, 2024–2033 (2009)

Leptin stimulates ovarian cancer cell growth and inhibits apoptosis by increasing cyclin D1 and Mcl-1 expression via the activation of the MEK/ERK1/2 and PI3K/Akt signaling pathways. Corrigendum in /10.3892/ijo.2016.3564

Obesity is known to be an important risk factor for many types of cancer, such as breast, prostate, liver and endometrial cancer. Recently, epidemiological studies have indicated that obesity correlates with an increased risk of developing ovarian cancer, the most lethal gynecological cancer in developed countries. Leptin is predominantly produced by adipocytes and acts as a growth factor and serum leptin levels positively correlate with the amount of body fat. In this study, we investigated the effects of leptin on the growth of ovarian cancer cells and the underlying mechanism(s) of action. Our results showed that leptin stimulated the growth of the OVCAR-3 ovarian cancer cell line using MTT assay and trypan blue exclusion. Using western blot analysis, we found that leptin enhanced the expression of cyclin D1 and Mcl-1, which are important regulators of cell proliferation and the inhibition of apoptosis. To investigate the signaling pathways that mediate the effects of leptin, cells were treated with leptin plus specific inhibitors of JAK2, PI3K/Akt and MEK/ERK1/2 and analysis of the phosphorylation state of proteins was carried out by western blot assays. We showed that the activation of the MEK/ERK1/2 and PI3K/Akt signaling pathways were involved in the growth-stimulating effect of leptin on ovarian cancer cell growth and the specific inhibitors of PI3K/Akt and MEK/ERK1/2 revealed that these two pathways interacted with each other. Our data demonstrate that leptin upregulates the expression of cyclin D1 and Mcl-1 to stimulate cell growth by activating the PI3K/Akt and MEK/ERK1/2 pathways in ovarian cancer.

Expression of a novel zinc-finger cDNA, IA-1, is associated with rat AR42J cells differentiation into insulin-positive cells.

Introduction IA-1, an insulinoma-associated cDNA-1, encodes a zinc-finger DNA-binding protein originally isolated from a human insulinoma subtraction library. Aim To demonstrate the restriction of IA-1 gene expression in human fetal pancreata of different gestational stages and to determine whether the expression of IA-1 gene is associated with rat AR42J cell differentiation into insulin-positive cells. Methodology To examine whether the IA-1 gene is associated with pancreatic endocrine cell differentiation, we used a rat pancreatic amphicrine cell line, AR42J, to investigate whether the expression of the IA-1 gene coincides with AR42J cells converting into either endocrine or exocrine lineage. We also examined a set of islet transcription factors that regulate key differentiation steps involved in activating the genes that confer the specialized functions of terminally differentiated pancreatic islet cells. Results When the AR42J cells were converted into insulin-positive cells induced by GLP-1, insulinoma conditioned-medium, or both, we observed a significant elevated expression of mRNA for IA-1 and islet-specific transcription factors such as Pdx-1, NeuroD/&bgr;2, and Nkx6.1. In contrast, dramatically decreased expression of mRNA for IA-1 and islet-specific transcription factors was displayed when AR42J cells were converted into the acinar-like phenotype by dexamethasone. Conclusions IA-1 gene was shown to be developmentally regulated in fetal pancreatic cells, and its expression pattern is consistent with parallel changes in islet-specific transcription factors during the endocrine differentiation of AR42J cells.

Zinc Finger Transcription Factor INSM1 Interrupts Cyclin D1 and CDK4 Binding and Induces Cell Cycle Arrest

INSM1 is a zinc finger transcription factor that plays an important role in pancreatic β-cell development. To further evaluate its role in cell fate determination, we investigated INSM1 effects on cell cycle function. The cyclin box of cyclin D1 is essential for INSM1 binding. Competitive pull-down and co-immunoprecipitation revealed that INSM1 binding to cyclin D1 interrupts its association with CDK4 and induces hypophosphorylation of the retinoblastoma protein. An inducible Tet-on system was established in Cos-7 and Panc-1 cells. Using serum starvation, we synchronized the cell cycle and subsequently induced cell cycle progression by serum stimulation. Comparison of the INSM1 induction group with the noninduced control group, INSM1 ectopic expression causes cell cycle arrest, whereas the INSM1-mediated cell cycle arrest could be reversed by cyclin D1 and CDK4 overexpression. The proline-rich N-terminal portion of INSM1 is required for cyclin D1 binding. Mutation of proline residues abolished cyclin D1 binding and also diminished its ability to induce cell cycle arrest. Cellular proliferation of Panc-1 cells was inhibited by INSM1 overexpression demonstrated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, soft agar colony formation, as well as tumor growth in a nude mouse model. Taken together, we provide evidence to support that INSM1 binds to cyclin D1, interrupts cell cycle signaling, and inhibits cellular proliferation.

Islet cell autoimmunity in youth onset diabetes mellitus in Northern India

We characterised a consecutive cohort of 132 youth onset diabetic individuals (age at onset<30 years, mean duration of disease 5.5+/-6.0 years) from North India, by serological determination of the determination of the islet cell autoantibodies, GAD(65) and IA2, and clinically for coexisting autoimmune thyroid disease, malnutrition and pancreatic calcification. Five types of diabetes were delineated: Type 1 (37%), ketosis resistant (32%), Type 2 (13%), fibrocalculous pancreatopathy (11%) and autoimmune polyglandular syndrome (7%). C-peptide response to glucagon was assessed in a representative subset of 50 patients with Type 1, ketosis resistant, and autoimmune polyglandular syndrome. A total of 22.4% of Type 1 and 30% of autoimmune polyglandular syndrome subjects showed both GAD(65) plus IA-2 autoantibody positivity, significantly more than the 4.7% positivity shown by the ketosis resistant type. However, GAD(65) antibody positivity alone was seen in 38% of ketosis resistant subjects which was significantly more than the 14.2 and 10% positivity seen in Type 1 and autoimmune polyglandular groups, respectively. The fibrocalculous pancreatopathy group showed GAD(65) plus IA-2 autoantibody positivity in 14.2% and GAD(65) autoantibody alone positivity in 7.1%. 26 and 60%, respectively, of the Type 1 and autoimmune polyglandular syndrome groups had thyroid microsomal autoantibody positivity. Type 1 showed significantly less C-peptide response to glucagon when compared to the ketosis resistant and autoimmune polyglandular syndrome groups. The controls and Type 2 diabetic individuals tested negative for islet cell autoimmunity markers. These findings demonstrate a role of islet cell autoimmunity in the pathogenesis of four out of the five clinical types of youth onset diabetes seen in North India.

Insulinoma-Associated Antigen-1 Zinc-Finger Transcription Factor Promotes Pancreatic Duct Cell Trans-Differentiation

Insulinoma-associated antigen-1 (INSM1/IA-1) is a unique zinc-finger transcription factor restrictedly expressed in pancreatic beta-cells during early pancreas development. INSM1 is transiently activated by the islet-specific endocrine factor neurogenin 3, and it subsequently regulates downstream target genes NeuroD1 and insulin during beta-cell maturation. Here, we examined how the INSM1 transcription factor contributes to endocrine cell differentiation using a defined serum-free medium-primed pancreatic duct cell model. We showed that ectopic expression of INSM1 can promote Panc-1 cell trans-differentiation. INSM1 up-regulates two islet transcription factors (ITFs), paired box 6 and homeodomain transcription factor 6.1, whereas other ITFs, including pancreatic duodenal homeobox-1 (Pdx-1), homeodomain transcription factor 2.2, NeuroD1, paired box 4, and neurogenin 3, were either down-regulated or absent. The result suggests that INSM1 is capable of regulating multiple ITFs and the insulin gene either directly or indirectly. When we overexpressed three ITFs, INSM1/Pdx-1/NeuroD1, in the Panc-1 differentiation model, higher insulin expression was observed in parallel with the activation of an additional ITF, neurogenin 3, signifying endocrine cell activation. Insulin expression from the three ITFs stimulation was readily detected by immunostaining and increased 40% as compared with the insulin-transferrin-selenium-LacZ control. Furthermore, we examined the differential chromatin acetylation patterns within the insulin promoter region using the chromatin immunoprecipitation assay. INSM1 alone can selectively enhance acetylation of histone H4, whereas NeuroD1 and Pdx-1 favor the acetylation of histone H3. Both H3 and H4 histone acetylations facilitate insulin gene expression. The consistent functional effect of INSM1, either with or without other ITFs, promotes pancreatic duct cell differentiation as well as induces Panc-1 cell cycle arrest.