Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer lacking effective drugs and therefore new treatment targets are needed. In this study, we define the role of homeobox protein B6 (HOXB6) and HOXB8 in controlling pancreatic cancer tumorigenesis and immune response. Transcriptomic analysis comparing human embryonic and PDAC tissue identified a large overlap of expression profiles suggesting a re-initiation of developmental programs in pancreatic cancer. Specifically, we identified the transcription factors HOXB6 and HOXB8 as potential regulators in PDAC. We described their functions in pancreatic cancer by performing transcriptomic and tumor tissue microarray analyses, in vitro assays in pancreatic and lung cancer cell lines and co-culture experiments with immune cells. Loss of HOXB6 and HOXB8 in pancreatic cancer cells inhibited cell proliferation, induced apoptosis and senescence and enhanced gemcitabine sensitivity. Moreover, reduced HOXB6 and HOXB8 expression in pancreatic and lung adenocarcinoma cell lines affected transcription of immune response pathways which resulted in an increased sensitivity of cancer cells to anti-tumorigenic activities of macrophages suggesting that the HOXB6 and HOXB8 immune regulatory function is conserved in different cancer types. Additionally, naïve M0 macrophages exposed to HOXB8 deficient PDAC cells were unable to differentiate into tumor-associated macrophages, suggesting that HOXB8 promotes the transition of initial anti-tumor macrophage to a tumor-promoting macrophage phenotype in pancreatic cancer. Our findings indicate that HOXB6 and HOXB8 play important roles in regulating cell proliferation, immune response, and treatment resistance to promote pancreatic cancer tumorigenesis and could be useful therapeutic targets.

Disruptions in circadian rhythm, partly controlled by the hormone melatonin, increase the risk of type 2 diabetes (T2D). Accordingly, a variant of the gene encoding the melatonin receptor 1B (MTNR1B) is robustly associated with increased risk of T2D. This single-nucleotide polymorphism (SNP; rs10830963; G-allele) is an expression quantitative trait locus (eQTL) in human pancreatic islets, conferring increased expression of MTNR1B, which is thought to perturb pancreatic β-cell function. To understand this pathogenic mechanism in detail, we utilized human induced pluripotent stem cells (hiPSC), derived from individuals with T2D carrying the MTNR1B G-allele. Patient-derived fibroblasts were reprogrammed to hiPSC and single-base genome editing by CRISPR/Cas9 was employed to create isogenic lines of either the C/C or G/G genotypes (nonrisk and risk, respectively). In addition, the human embryonic stem cell (hESC) line (HUES4) was subjected to genome editing to create isogenic lines of either the C/C or G/G genotypes. hiPSC and hESC were differentiated into β-like cells, using a 50-day 2D protocol. Single-base genome editing generated cells with the desired genotype at a success rate of > 90%. Expression of stage-specific markers confirmed differentiation of both hiPSC and hESC into β-cells. MTNR1B mRNA levels were consistently low in differentiated β-cells, precluding quantitative analysis of gene expression. Western blot analyses indicated slightly higher levels of the MTNR1B protein in differentiated β-cells carrying the risk allele, which is in accord with the notion that rs10830963 (G-allele) functions as an eQTL in β-cells. Insulin secretion in response to the combination of high glucose and IBMX was comparable between genotypes, whereas the addition of melatonin appeared to reduce insulin secretion more efficiently in cells carrying the G-allele. While our data suggest elevated MTNR1B protein levels in stem cell-derived β-like cells carrying the risk allele, these cells do not appear to be sufficiently mature to establish rs10830963 as an eQTL at the mRNA level. The observed nominal increase in melatonin sensitivity in G-allele-carrying cells is suggestive of a functional contribution of rs10830963 to β-cell dysfunction; however, this interpretation remains tentative and will require further validation in more mature β-cell models.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer lacking effective drugs and therefore new treatment targets are needed. Transcriptomic analysis comparing human embryonic and PDAC tissue identified a large overlap of expression profiles suggesting a re-initiation of developmental programs in pancreatic cancer. Specifically, we identified the transcription factors HOXB6 and HOXB8 as potential key regulators in PDAC. Loss of HOXB6 and HOXB8 in pancreatic cancer cells inhibited cell proliferation, induced apoptosis and senescence and enhanced gemcitabine sensitivity. Moreover, reduced HOXB6 and HOXB8 expression in pancreatic and lung adenocarcinoma cell lines affected transcription of immune response pathways which resulted in an increased sensitivity of cancer cells to anti-tumorigenic activities of macrophages suggesting that the HOXB6 and HOXB8 immune regulatory pattern is conserved in different cancer types. Additionally, naïve M0 macrophages exposed to HOXB8 deficient PDAC cells were unable to differentiate into tumor associated macrophages, suggesting that HOXB8 promotes the transition of initial anti-tumor macrophage to a tumor-promoting macrophage phenotype in pancreatic cancer. Our findings indicate that HOXB6 and HOXB8 play important roles in regulating cell proliferation, immune response and treatment resistance to promote pancreatic cancer tumorigenesis and could be useful therapeutic targets. ### Competing Interest Statement The authors have declared no competing interest.

Graves' disease (GD) and Graves' ophthalmopathy (GO) are complex autoimmune diseases. This study delved into the impact of cigarette smoke extract (CSE), simvastatin, and/or diclofenac on peripheral blood mononuclear cells (PBMCs). Specifically, we explored alterations in IL-1B, IL-6, PTGS2 expression, B- and T-lymphocyte proliferation, and Immunoglobulin G (IgG) production. We also assessed IGF1's influence on B- and T-lymphocyte proliferation. PBMCs from Graves' patients were exposed to CSE with/without simvastatin and/or diclofenac. Gene and protein expression was compared with untreated PBMCs. B- and T-lymphocyte proliferation was assessed following IGF1 treatment. PBMCs exposed to CSE exhibited increased expression of IL-1B (6-fold), IL-6 (10-fold), and PTGS2 (5.6-fold), and protein levels of IL-1B (4-fold), IL-6 (16-fold) and PGE2 (3.7-fold) compared with untreated PBMCs. Simvastatin and/or diclofenac downregulated the expression of PTGS2 (0.5-fold), IL-6 (0.4-fold), and IL-1B (0.6-fold), and the protein levels of IL-1B (0.6-fold), IL-6 (0.6-fold), and PGE2 (0.6-fold) compared with untreated PBMCs. CSE exposure in PBMCs increased the proliferation of B and T lymphocytes by 1.3-fold and 1.4-fold, respectively, compared with untreated. CSE exposure increased IgG (1.5-fold) in supernatant from PBMCs isolated from Graves' patients. IGF1 treatment increased the proliferation of B and T lymphocytes by 1.6-fold. Simvastatin downregulated the proliferation of B and T lymphocytes by 0.7-fold. Our study shows that CSE significantly upregulated the expression and release of the inflammatory markers PTGS2, IL-6 and IL-1B,the IgG levels, and the proliferation of B and T lymphocytes. Additionally, IGF1 increased the proliferation of B and T lymphocytes. Finally, these effects were decreased by diclofenac and/or simvastatin treatment.

ABSTRACT Hormone secretion from pancreatic islets is essential for glucose homeostasis, and loss or dysfunction of islet cells is a hallmark of type 2 diabetes. Maf transcription factors are crucial for establishing and maintaining adult endocrine cell function. However, during pancreas development, MafB is not only expressed in insulin- and glucagon-producing cells, but also in Neurog3+ endocrine progenitor cells, suggesting additional functions in cell differentiation and islet formation. Here, we report that MafB deficiency impairs β cell clustering and islet formation, but also coincides with loss of neurotransmitter and axon guidance receptor gene expression. Moreover, the observed loss of nicotinic receptor gene expression in human and mouse β cells implied that signaling through these receptors contributes to islet cell migration/formation. Inhibition of nicotinic receptor activity resulted in reduced β cell migration towards autonomic nerves and impaired β cell clustering. These findings highlight a novel function of MafB in controlling neuronal-directed signaling events required for islet formation.

Reduced expression of exocytotic genes is associated with functional defects in insulin exocytosis contributing to impaired insulin secretion and type 2 diabetes (T2D) development. MAFA and MAFB transcription factors regulate β‐cell physiology, and their gene expression is reduced in T2D β cells. We investigate if loss of MAFA and MAFB in human β cells contributes to T2D progression by regulating genes required for insulin exocytosis.

The amplification of glucose-stimulated insulin secretion (GSIS) through incretin signaling is critical for maintaining physiological glucose levels. Incretins, like glucagon-like peptide 1 (GLP1), are a target of type 2 diabetes drugs aiming to enhance insulin secretion. Here we show that the protein phosphatase 1 inhibitor protein 1A (PPP1R1A), is expressed in β-cells and that its expression is reduced in dysfunctional β-cells lacking MafA and upon acute MafA knock down. MafA is a central regulator of GSIS and β-cell function. We observed a strong correlation of MAFA and PPP1R1A mRNA levels in human islets, moreover, PPP1R1A mRNA levels were reduced in type 2 diabetic islets and positively correlated with GLP1-mediated GSIS amplification. PPP1R1A silencing in INS1 (832/13) β-cells impaired GSIS amplification, PKA-target protein phosphorylation, mitochondrial coupling efficiency and also the expression of critical β-cell marker genes like MafA, Pdx1, NeuroD1 and Pax6. Our results demonstrate that the β-cell transcription factor MafA is required for PPP1R1A expression and that reduced β-cell PPP1R1A levels impaired β-cell function and contributed to β-cell dedifferentiation during type 2 diabetes. Loss of PPP1R1A in type 2 diabetic β-cells may explains the unresponsiveness of type 2 diabetic patients to GLP1R-based treatments.

Type 2 diabetes, characterized by dysfunction of pancreatic β-cells and insulin resistance in peripheral organs, accounts for more than 90% of all diabetes. Despite current developments of new drugs and strategies to prevent/treat diabetes, there is no ideal therapy targeting all aspects of the disease. Restoration, however, of insulin-producing β-cells, as well as insulin-responsive cells, would be a logical strategy for the treatment of diabetes. In recent years, generation of transplantable cells derived from stem cells in vitro has emerged as an important research area. Pluripotent stem cells, either embryonic or induced, are alternative and feasible sources of insulin-secreting and glucose-responsive cells. This notwithstanding, consistent generation of robust glucose/insulin-responsive cells remains challenging. In this review, we describe basic concepts of the generation of induced pluripotent stem cells and subsequent differentiation of these into pancreatic β-like cells, myotubes, as well as adipocyte- and hepatocyte-like cells. Use of these for modeling of human disease is now feasible, while development of replacement therapies requires continued efforts.

The aquaglyceroporins are a subfamily of aquaporins that conduct both water and glycerol. Aquaporin-3 (AQP3) has an important physiological function in renal water reabsorption, and AQP3-mediated hydrogen peroxide (H2O2) permeability can enhance cytokine signaling in several cell types. The related aquaglyceroporin AQP7 is required for dendritic cell chemokine responses and antigen uptake. Selective small-molecule inhibitors are desirable tools for investigating the biological and pathological roles of these and other AQP isoforms. Here, using a calcein fluorescence quenching assay, we screened a library of 7360 drug-like small molecules for inhibition of mouse AQP3 water permeability. Hit confirmation and expansion with commercially available substances identified the ortho-chloride-containing compound DFP00173, which inhibited mouse and human AQP3 with an IC50 of approximate to 0.1-0.4 m but had low efficacy toward mouse AQP7 and AQP9. Surprisingly, inhibitor specificity testing revealed that the methylurea-linked compound Z433927330, a partial AQP3 inhibitor (IC50, approximate to 0.7-0.9 m), is a potent and efficacious inhibitor of mouse AQP7 water permeability (IC50, approximate to 0.2 m). Stopped-flow light scattering measurements confirmed that DFP00173 and Z433927330 inhibit AQP3 glycerol permeability in human erythrocytes. Moreover, DFP00173, Z433927330, and the previously identified AQP9 inhibitor RF03176 blocked aquaglyceroporin H2O2 permeability. Molecular docking to AQP3, AQP7, and AQP9 homology models suggested interactions between these inhibitors and aquaglyceroporins at similar binding sites. DFP00173 and Z433927330 constitute selective and potent AQP3 and AQP7 inhibitors, respectively, and contribute to a set of isoform-specific aquaglyceroporin inhibitors that will facilitate the evaluation of these AQP isoforms as drug targets.

Voltage-gated Ca2+ (CaV) channels trigger glucose-induced insulin secretion in pancreatic beta-cell and their dysfunction increases diabetes risk. These heteromeric complexes include the main subunit alpha1, and the accessory ones, including subunit gamma that remains unexplored. Here, we demonstrate that CaV gamma subunit 4 (CaVγ4) is downregulated in islets from human donors with diabetes, diabetic Goto-Kakizaki (GK) rats, as well as under conditions of gluco-/lipotoxic stress. Reduction of CaVγ4 expression results in decreased expression of L-type CaV1.2 and CaV1.3, thereby suppressing voltage-gated Ca2+ entry and glucose stimulated insulin exocytosis. The most important finding is that CaVγ4 expression is controlled by the transcription factor responsible for beta-cell specification, MafA, as verified by chromatin immunoprecipitation and experiments in beta-cell specific MafA knockout mice (MafA Δβcell ). Taken together, these findings suggest that CaVγ4 is necessary for maintaining a functional differentiated beta-cell phenotype. Treatment aiming at restoring CaVγ4 may help to restore beta-cell function in diabetes.

Maf transcription factors are critical regulators of beta-cell function. We have previously shown that reduced MafA expression in human and mouse islets is associated with a pro-inflammatory gene signature. Here, we investigate if the loss of Maf transcription factors induced autoimmune processes in the pancreas. Transcriptomics analysis showed expression of pro-inflammatory as well as immune cell marker genes. However, clusters of CD4+ T and B220+ B cells were associated primarily with adult MafA −/− MafB +/− , but not MafA −/− islets. MafA expression was detected in the thymus, lymph nodes and bone marrow suggesting a novel role of MafA in regulating immune-cell function. Analysis of pancreatic lymph node cells showed activation of CD4+ T cells, but lack of CD8+ T cell activation which also coincided with an enrichment of naïve CD8+ T cells. Further analysis of T cell marker genes revealed a reduction of T cell receptor signaling gene expression in CD8, but not in CD4+ T cells, which was accompanied with a defect in early T cell receptor signaling in mutant CD8+ T cells. These results suggest that loss of MafA impairs both beta- and T cell function affecting the balance of peripheral immune responses against islet autoantigens, resulting in local inflammation in pancreatic islets.

Type 1 (T1D) and type 2 (T2D) diabetes are triggered by a combination of environmental and/or genetic factors. Maf transcription factors regulate pancreatic beta (β)-cell function, and have also been implicated in the regulation of immunomodulatory cytokines like interferon-β (IFNβ1). In this study, we assessed MAFA and MAFB co-expression with pro-inflammatory cytokine signaling genes in RNA-seq data from human pancreatic islets. Interestingly, MAFA expression was strongly negatively correlated with cytokine-induced signaling (such as IFNAR1, DDX58) and T1D susceptibility genes (IFIH1), whereas correlation of these genes with MAFB was weaker. In order to evaluate if the loss of MafA altered the immune status of islets, MafA deficient mouse islets (MafA−/−) were assessed for inherent anti-viral response and susceptibility to enterovirus infection. MafA deficient mouse islets had elevated basal levels of Ifnβ1, Rig1 (DDX58 in humans), and Mda5 (IFIH1) which resulted in reduced virus propagation in response to coxsackievirus B3 (CVB3) infection. Moreover, an acute knockdown of MafA in β-cell lines also enhanced Rig1 and Mda5 protein levels. Our results suggest that precise regulation of MAFA levels is critical for islet cell-specific cytokine production, which is a critical parameter for the inflammatory status of pancreatic islets.

Vitamin A-derived retinoic acid (RA) signals are critical for the development of several organs, including the pancreas. However, the tissue-specific control of RA synthesis in organ and cell lineage development has only poorly been addressed in vivo. Here, we show that retinol dehydrogenase-10 (Rdh10), a key enzyme in embryonic RA production, has important functions in pancreas organogenesis and endocrine cell differentiation. Rdh10 was expressed in the developing pancreas epithelium and surrounding mesenchyme. Rdh10 null mutant mouse embryos exhibited dorsal pancreas agenesis and a hypoplastic ventral pancreas with retarded tubulogenesis and branching. Conditional disruption of Rdh10 from the endoderm caused increased mortality, reduced body weight, and lowered blood glucose levels after birth. Endodermal Rdh10 deficiency led to a smaller dorsal pancreas with a reduced density of early glucagon+ and insulin+ cells. During the secondary transition, the reduction of Neurogenin3+ endocrine progenitors in the mutant dorsal pancreas accounted for fewer α- and β-cells. Changes in the expression of α- and β-cell-specific transcription factors indicated that Rdh10 might also participate in the terminal differentiation of endocrine cells. Together, our results highlight the importance of both mesenchymal and epithelial Rdh10 for pancreogenesis and the first wave of endocrine cell differentiation. We further propose a model in which the Rdh10-expressing exocrine tissue acts as an essential source of RA signals in the second wave of endocrine cell differentiation.

Monoamine and acetylcholine neurotransmitters from the autonomic nervous system (ANS) regulate insulin secretion in pancreatic islets. The molecular mechanisms controlling neurotransmitter signaling in islet β cells and their impact on diabetes development are only partially understood. Using a glucose-intolerant, MafA-deficient mouse model, we demonstrate that MAFA controls ANS-mediated insulin secretion by activating the transcription of nicotinic (ChrnB2 and ChrnB4) and adrenergic (Adra2A) receptor genes, which are integral parts of acetylcholine- and monoamine-signaling pathways. We show that acetylcholine-mediated insulin secretion requires nicotinic signaling and that nicotinic receptor expression is positively correlated with insulin secretion and glycemic control in human donor islets. Moreover, polymorphisms spanning MAFA-binding regions within the human CHRNB4 gene are associated with type 2 diabetes. Our data show that MAFA transcriptional activity is required for establishing β cell sensitivity to neurotransmitter signaling and identify nicotinic signaling as a modulator of insulin secretion impaired in type 2 diabetes.