Pancreatic Ductal Adenocarcinoma (PDAC) circulating tumor cells influence myeloid cell differentiation to support their survival and immunoresistance in portal vein circulation.

The portal venous circulation provides a conduit for pancreatic ductal adenocarcinoma (PDAC) tumor cells to the liver parenchyma sinusoids, a frequent site of metastasis. Turbulent flow in the portal circulation promotes retention of PDAC shed circulating tumor cells (CTC) and myeloid-derived immunosuppressor cells (MDSC). Excessive colony stimulating factor-1 receptor (CSF1R) signaling can induce myeloid differentiation to MDSC and transformation of MDSC to myeloid-derived fibroblasts (M-FB). Interactions between PDAC CTC and M-FB in the portal blood promotes the formation of immunoresistant clusters that enhance CTC proliferation, migration, and survival. Analysis of portal and peripheral blood samples collected intraoperatively from 30 PDAC patients undergoing pancreatico-duodenectomy showed that PDAC patient plasma contained high levels of macrophage colony stimulating factor (M-CSF/CSF1), granulocyte-macrophage colony stimulating factor (GM-CSF/CSF2), interleukin-8 (IL-8), and interleukin-34 (IL-34) compared to healthy control levels. Moreover, the level of M-CSF in portal blood was significantly higher than that detected in the peripheral blood of PDAC patients. PDAC CTC aseptically isolated by fluorescence activated cell sorting (FACS) out of freshly collected patient portal blood mononuclear cells (PortalBMC) had elevated RNA expression of IL34 (IL-34 gene) and CSF1 (M-CSF/CSF1 gene) which both signal through CSF1R. PDAC CTC also had high levels of RNA expression for CXCL8, the gene encoding chemokine interleukin-8 (IL-8) which can attract myeloid cells through their CXCR2 receptors. FACS-isolated portal PDAC CTC and M-FB co-cultured ex vivo had increased CTC proliferation, motility, and cluster formation compared to CTC cultured alone. CSF1R and CXCR2 cell surface expression were found on PDAC portal blood CTC and M-FB, suggesting that both cell types may respond to M-CSF, IL-34, and IL-8-mediated signaling. Portal PDAC CTC displayed enhanced RNA expression of CSF1 and IL34, while CTC+M-FB+ clusters formed in vivo had increased RNA expression of CSF2 and IL34. Portal M-FB were found to have high CSF1R RNA expression. CTC isolated from ex vivo 7-day cultures of PDAC patient portal blood mononuclear cells (PortalBMC) expressed elevated CSF1, IL34, and IL8 RNA, and CSF1 expression was elevated in M-FB. Treatment with rabbit anti-CSF1R antibodies decreased CTC proliferation. Treatment of PortalBMC cultures with humanized anti-CSF1R, humanized anti-IL-8, or anti-IL-34 antibodies disrupted CTC cluster formation and increased CTC apoptosis. U937 myeloid precursor cell line cultures treated with conditioned media from PortalBMC ex vivo cultures without treatment or treated with anti-IL-8 and/or anti-CSF1R did not prevent myeloid differentiation in the myeloid precursor cell line U937 to macrophage, dendritic cell, MDSC, and M-FB phenotypes; whereas, U937 cultures treated with conditioned media from PortalBMC ex vivo cultures exposed to anti-IL-34 were significantly inhibited in their myeloid differentiation to all but the M-FB phenotype. PDAC patient T cells that were found phenotypically anergic (CD3+CD25+CTLA4+PD1L1+) in PortalBMC could be re-activated (CD3+CD25+CTLA4-PD1L1-), and displayed increased interferon gamma (IFNγ) production when PortalBMC ex vivo cultures were treated with anti-CSF1R, anti-IL-8, and anti-IL-34 antibodies alone or in combination. These findings suggest that PDAC CTC have the potential to influence myeloid differentiation and/or antigen presenting cell activation in the PDAC portal blood microenvironment, and that disruption of CTC/M-FB interactions may be potential targets for reversing the immunosuppression supporting CTC survival in the portal blood.

Discovery of Molecular Interactions of the Human Melanocortin-4 Receptor (hMC4R) Asp189 (D189) Amino Acid with the Endogenous G-Protein-Coupled Receptor (GPCR) Antagonist Agouti-Related Protein (AGRP) Provides Insights to AGRP's Inverse Agonist Pharmacology at the hMC4R.

The melanocortin receptors (MCRs) are important for numerous biological pathways, including feeding behavior and energy homeostasis. In addition to endogenous peptide agonists, this receptor family has two naturally occurring endogenous antagonists, agouti and agouti-related protein (AGRP). At the melanocortin-4 receptor (MC4R), the AGRP ligand functions as an endogenous inverse agonist in the absence of agonist and as a competitive antagonist in the presence of agonist. At the melanocortin-3 receptor (MC3R), AGRP functions solely as a competitive antagonist in the presence of agonist. The molecular interactions that differentiate AGRP's inverse agonist activity at the MC4R have remained elusive until the findings reported herein. Upon the basis of homology molecular modeling approaches, we previously postulated a unique interaction between the D189 position of the hMC4R and Asn114 of AGRP. To further test this hypothesis, six D189 mutant hMC4Rs (D189A, D189E, D189N, D189Q, D189S, and D189K) were generated and pharmacologically characterized resulting in the discovery of differences in inverse agonist activity of AGRP and an 11 macrocyclic compound library. These data support the hypothesized interaction between the hMC4R D189 position and Asn114 residue of AGRP and define critical ligand-receptor molecular interactions responsible for the inverse agonist activity of AGRP at the hMC4R.

Identification of a novel IL-5 signaling pathway in chronic pancreatitis and crosstalk with pancreatic tumor cells.

BACKGROUND: While inflammation is associated with pancreatic cancer, the underlying mechanisms leading to cancer initiation are still being delineated. Eosinophils may promote or inhibit tumor growth, although the specific role in pancreatic cancer has yet to be determined. Eosinophil-supporting cytokine interleukin-5 and receptor are likely to have a role, but the significance in the pancreatic cancer microenvironment is unknown. METHODS: Genetically engineered Akt1Myr/KRasG12D and KRasG12D mice were used to model changes induced by chronic inflammation. Tissue samples were collected to analyze the tumor microenvironment and infiltration of immune cells, whereas serum was collected to analyze cytokine and amylase activity in the inflammatory model. The expression of IL-5R and the effects of IL-5 were analyzed in human and murine tumor cells. RESULTS: Compound Akt1Myr/KRasG12D mice, compared to single KRasG12D or Akt1Myr mice, exhibited increased tissue damage after repeat inductions of inflammation, and had accelerated tumor development and metastasis. M2 macrophages and newly identified eosinophils co-localized with fibrotic regions rather than infiltrating into tumors, consistent with immune cell privilege. The majority of eosinophils found in the pancreas of Akt1Myr/KRasG12D mice with chronic inflammation lacked the cytotoxic NKG2D marker. IL-5 expression was upregulated in pancreatic cells in response to inflammation, and then diminished in advanced lesions. Although not previously described in pancreatic tumors, IL-5Rα was increased during mouse pancreatic tumor progression and expressed in human pancreatic ductal adenocarcinomas (7 of 7 by immunohistochemistry). IL-5 stimulated tumor cell migration and activation through STAT5 signaling, thereby suggesting an unreported tumor-promoting role for IL-5Rα in pancreatic cancer. CONCLUSIONS: Chronic inflammation induces increased pancreatic cancer progression and immune cells such as eosinophils are attracted to areas of fibrosis. Results suggest that IL-5 in the pancreatic compartment stimulates increased IL-5Rα on ductal tumor cells to increase pancreatic tumor motility. Collectively, IL-5/IL-5Rα signaling in the mouse and human pancreatic tumors microenvironment is a novel mechanism to facilitate tumor progression. Additional file 1: Video Abstract.

K-RAS Mutant Gene Found in Pancreatic Juice Activated Chromatin From Peri-ampullary Adenocarcinomas.

External pancreatic duct stents inserted after resection of pancreatic head tumors provide unique access to pancreatic juice analysis of genetic and metabolic components that may be associated with peri-ampullary tumor progression. For this pilot study, portal venous blood and pancreatic juice samples were collected from 17 patients who underwent pancreaticoduodenectomy for peri-ampullary tumors. Portal vein circulating tumor cells (CTC) were isolated by high-speed fluorescence-activated cell sorting (FACS) and analyzed by quantitative reverse transcription polymerase chain reaction (RT-PCR) for K-RAS exon 12 mutant gene expression (K-RASmut). DNA, chromatin, and histone acetylated active chromatin were isolated from pancreatic juice samples by chromatin immunoprecipitation (ChIP) and the presence of K-RASmut and other cancer-related gene sequences detected by quantitative polymerase chain reaction (PCR) and ChIP-Seq. Mutated K-RAS gene was detectable in activated chromatin in pancreatic juice secreted after surgical resection of pancreatic, ampullary and bile duct carcinomas and directly correlated with the number of CTC found in the portal venous blood (P = .0453). ChIP and ChIP-Seq detected acetylated chromatin in peri-ampullary cancer patient juice containing candidate chromatin loci, including RET proto-oncogene, not found in similar analysis of pancreatic juice from non-malignant ampullary adenoma. The presence of active tumor cell chromatin in pancreatic juice after surgical removal of the primary tumor suggests that viable cancer cells either remain or re-emerge from the remnant pancreatic duct, providing a potential source for tumor recurrence and cancer relapse. Therefore, epigenetic analysis for active chromatin in pancreatic juice and portal venous blood CTC may be useful for prognostic risk stratification and potential identification of molecular targets in peri-ampullary cancers.

Pancreatic and bile duct cancer circulating tumor cells (CTC) form immune-resistant multi-cell type clusters in the portal venous circulation.

Circulating tumor cells (CTC) enter the blood from many carcinomas and represent a likely source of metastatic dissemination. In contrast to the peripheral circulation, KRAS mutation- positive CTC thrive in the portal venous blood of patients with pancreatic ductal adenocarcinoma (PDAC). To analyze the essential interactions that contribute to carcinoma CTC growth and immune resistance, portal venous blood was collected during pancreatico-duodenectomy in 41 patients with peri-ampullary pathologies (PDAC = 11; ampullary adenocarcinoma (AA) = 15; distal cholangiocarcinoma (CC) = 6; IPMN = 7; non-malignant pancreatitis = 2). FACS-isolated cell populations from the portal circulation were reconstituted ex vivo using mixed cell reaction cultures (MCR). During the first 48hr, PDAC, AA, and CC patient CTC were all highly proliferative (mean 1.7 hr/cell cycle, 61.5% ± 20% growing cells) and resistant to apoptosis (mean 39% ±  25% apoptotic cells). PDAC CTC proliferation and resistance to T cell cytotoxicity were decreased among patients who received pre-operative chemotherapy (p = 0.0019, p = 0.0191, respectively). After 7 days in culture, CTC from PDAC, CC, and AA patients recruited multiple immune cell types, including CD105 + CD14 + myeloid fibroblasts, to organize into spheroid-like clusters. It was only in PDAC and CC-derived MCR that cluster formation promoted CTC survival, growth, and fibroblast differentiation. FACS depletion of CTC or myeloid fibroblast cells eliminated cluster network formation, and re-introduction of these cell populations reconstituted such ability. Our findings suggest that PDAC and CC CTC survival within the portal venous circulation is supported by their interactions with immune cells within multi-cell type clusters that could represent vectors of local recurrence and metastatic progression.

Portal Venous Blood Circulation Supports Immunosuppressive Environment and Pancreatic Cancer Circulating Tumor Cell Activation.

OBJECTIVES: Aggressive spread and liver metastases are predominant features of pancreatic ductal adenocarcinoma (PDAC). This study investigates activation of PDAC circulating tumor cells (CTC) and immunosuppression in the portal venous system. METHODS: Portal venous and peripheral blood were collected during pancreaticoduodenectomy from patients with PDAC (n = 21) or other non-PDAC pancreatic conditions (n = 20). Circulating tumor cells were isolated by fluorescence-activated cell sorting and characterized for messenger RNA (mRNA) expression and acetylated chromatin encoding K-RAS exon 12 mutation (K-RASmut). Myeloid-derived suppressor cells (MDSC) were identified using flow cytometry. RESULTS: Pancreatic ductal adenocarcinoma K-RASmut mRNA expression in portal venous blood CTC was significantly elevated compared with preoperative and postoperative peripheral blood (P = 0.0123 and P = 0.0246, respectively). There was no significant variation in total CTC numbers between portal and peripheral blood.Portal venous M-MDSC were elevated compared with peripheral blood in PDAC patients (P = 0.0065). M-MDSC increases correlated with K-RASmut mRNA-expressing CTC present in PDAC portal blood (P < 0.0001). CONCLUSIONS: Association of MDSC with active CTC in portal venous blood may support immunosuppression within the portal venous circulation to promote PDAC CTC survival.

Blatchford Score Is Superior to AIMS65 Score in Predicting the Need for Clinical Interventions in Elderly Patients with Nonvariceal Upper Gastrointestinal Bleed.

Background. Blatchford and AIMS65 scores were developed to risk stratify patients with upper gastrointestinal bleed (UGIB). We sought to assess the performance of Blatchford and AIMS65 scores in predicting outcomes in elderly patients with nonvariceal UGIB. Methods. A retrospective cohort study of elderly patients (over 65 years of age) with nonvariceal UGIB admitted to a tertiary care center. Primary outcome was a combined outcome of in-hospital mortality, need for any therapeutic endoscopic, radiologic, or surgical intervention, rebleeding within 30 days, or blood transfusion. Secondary outcome was a combined outcome of in-hospital mortality or need for an intervention to control the bleed. Results. 164 patients were included. The primary outcome occurred in 119 (72.5%) patients. The secondary outcome occurred in 12 patients (7.2%). Blatchford score was superior to AIMS65 score in predicting the primary outcome (area under the receiver-operator curve (AUROC) 0.84 versus 0.68, resp., p < 0.001). Both scores performed poorly in predicting the secondary outcome (AUROC 0.56 versus 0.52, resp., p = 0.18). Conclusions. Blatchford score could be useful in predicting the need for hospital based interventions in elderly patients with nonvariceal UGIB. Blatchford and AIMS65 scores are poor predictors of the need for a therapeutic intervention to control bleeding.

Csf2 and Ptgs2 Epigenetic Dysregulation in Diabetes-prone Bicongenic B6.NODC11bxC1tb Mice.

In Type 1 diabetic (T1D) human monocytes, STAT5 aberrantly binds to epigenetic regulatory sites of two proinflammatory genes, CSF2 (encoding granulocyte-macrophage colony-stimulating factor) and PTGS2 (encoding prostaglandin synthase 2/cyclooxygenase 2). Bicongenic B6.NOD C11bxC1tb mice re-create this phenotype of T1D monocytes with only two nonobese diabetic (NOD) Idd subloci (130.8 Mb-149.7 Mb, of Idd5 on Chr 1 and 32.08-53.85 Mb of Idd4.3 on Chr11) on C57BL/6 genetic background. These two Idd loci interact through STAT5 binding at upstream regulatory regions affecting Csf2 (Chr 11) and Ptgs2 (Chr 1) expression. B6.NODC11bxC1tb mice exhibited hyperglycemia and immune destruction of pancreatic islets between 8 and 30 weeks of age, with 12%-22% penetrance. Thus, B6.NODC11bxC1tb mice embody NOD epigenetic dysregulation of gene expression in myeloid cells, and this defect appears to be sufficient to impart genetic susceptibility to diabetes in an otherwise genetically nonautoimmune mouse.

Persistent STAT5 phosphorylation and epigenetic dysregulation of GM-CSF and PGS2/COX2 expression in Type 1 diabetic human monocytes.

STAT5 proteins are adaptor proteins for histone acetylation enzymes. Histone acetylation at promoter and enhancer chromosomal regions opens the chromatin and allows access of transcription enzymes to specific genes in rapid response cell signals, such as in inflammation. Histone acetylation-mediated gene regulation is involved in expression of 2 key inflammatory response genes: CSF2, encoding granulocyte-macrophage colony stimulating factor (GM-CSF), and PTGS2, encoding prostaglandin synthase 2/cyclooxygenase 2 (PGS2/COX2). Prolonged CSF2 expression, high GM-CSF production, and GM-CSF activation of PTGS2 gene expression all are seen in type 1 diabetes (T1D) monocytes. Persistent phosphorylation activation of monocyte STAT5 (STAT5Ptyr) is also found in individuals with or at-risk for T1D. To examine whether elevated T1D monocyte STAT5Ptyr may be associated with aberrant inflammatory gene expression in T1D, blood monocytes from non-autoimmune controls and T1D patients were analyzed by flow cytometry for STAT5Ptyr activation, and by chromatin immuno-precipitation (ChIP) analyses for STAT5Ptyr's ability to bind at CSF2 and PTGS2 regulatory sites in association with histone acetylation. In unstimulated monocytes, STAT5Ptyr was elevated in 59.65% of T1D, but only 2.44% of control subjects (p<0.0001). Increased STAT5Ptyr correlated with T1D disease duration (p = 0.0030, r(2) = 0.0784). Unstimulated (p = 0.140) and GM-CSF-stimulated (p = 0.0485) T1D monocytes, had greater STAT5Ptyr binding to epigenetic regulatory sites upstream of CSF2 than control monocytes. Increased STAT5Ptyr binding in T1D monocytes was concurrent with binding at these sites of STAT6Ptyr (p = 0.0283), CBP/P300 histone acetylase, acetylated histones H3, SMRT/NCoR histone deacetylase (p = 0.0040), and RNA Polymerase II (p = 0.0040). Our study indicates that in T1D monocytes, STAT5Ptyr activation is significantly higher and that STAT5Ptyr is found bound to CSF2 promoter and PTGS2 enhancer regions coincident with histone acetylation and RNA polymerase II. These findings suggest that the persistent activation of STAT5 by GM-CSF may be involved in altering the epigenetic regulation of these inflammatory response genes in T1D monocytes.

In vitro generation of functional insulin-producing cells from human bone marrow-derived stem cells, but long-term culture running risk of malignant transformation.

Efforts involving therapeutic islet cell transplantation have been hampered by limited islet availability and immune rejection. In vitro transdifferentiation of human bone marrow-derived stem (hBMDS) cells into functional insulin-producing cells promises to provide a tissue source for autologous cell transplantation. In this study, we isolated hBMDS cells, developed a single-cell-derived stem cell line, and induced the cells to differentiate into islet-like clusters. These islet-like cells expressed multiple genes related to islet development and beta cell function (e.g., Pdx-1, Ngn-3, Islet-1, Neuro-D, Pax4, IAPP, and insulin) and produced insulin and C-peptide within these cells. These islet-like cells demonstrated time-dependent glucose-stimulated insulin release, and the ability to ameliorate hyperglycemia in chemically induced diabetic mice. However, these transplanted differentiated cells became tumorigenic in diabetic immunocompromised mice and their spontaneous transformation was confirmed by a marked increase in growth rate and inactivation of tumor suppressor genes (P21 and P16) by promoter hypermethylation. In conclusion, while hBMDS cells can be transdifferentiated into competent insulin-producing cells, and while such cell might be a potential source for autologous cell therapy for type 1 diabetes, caution is strongly advised in view of the neoplastic propensity of hBMDS cells, especially after a long-term culture in vitro.

Developing a nanoparticle test for prostate cancer scoring.

BACKGROUND: Over-diagnosis and treatment of prostate cancer has been a major problem in prostate cancer care and management. Currently the most relevant prognostic factor to predict a patient's risk of death due to prostate cancer is the Gleason score of the biopsied tissue samples. However, pathological analysis is subjective, and the Gleason score is only a qualitative estimate of the cancer malignancy. Molecular biomarkers and diagnostic tests that can accurately predict prostate tumor aggressiveness are rather limited. METHOD: We report here for the first time the development of a nanoparticle test that not only can distinguish prostate cancer from normal and benign conditions, but also has the potential to predict the aggressiveness of prostate cancer quantitatively. To conduct the test, a prostate tissue lysate sample is spiked into a blood serum or human IgG solution and the spiked sample is incubated with a citrate-protected gold nanoparticle solution. IgG is known to adsorb to citrate-protected gold nanoparticles to form a "protein corona" on the nanoparticle surface. From this study, we discovered that certain tumor-specific molecules can interact with IgG and change the adsorption behavior of IgG to the gold nanoparticles. This change is reflected in the nanoparticle size of the assay solution and detected by a dynamic light scattering technique. Assay data were analyzed by one-way ANOVA for multiple variant analysis, and using the Student t-test or nonparametric Mann-Whitney U-tests for pairwise analyses. RESULTS: An inverse, quantitative correlation of the average nanoparticle size of the assay solution with tumor status and histological diagnostic grading was observed from the nanoparticle test. IgG solutions spiked with prostate tumor tissue exhibit significantly smaller nanoparticle size than the solutions spiked with normal and benign tissues. The higher grade the tumor is, the smaller the nanoparticle size is. The test particularly revealed large differences among the intermediate Grade 2 tumors, and suggested the need to treat them differently. CONCLUSION: Development of a new nanoparticle test may provide a quantitative measure of the prostate cancer aggressiveness. If validated in a larger study of patients with prostate cancer, this test could become a new diagnostic tool in conjunction with Gleason Score pathology diagnostics to better distinguish aggressive cancer from indolent tumor.

Pharmacological characterization of 30 human melanocortin-4 receptor polymorphisms with the endogenous proopiomelanocortin-derived agonists, synthetic agonists, and the endogenous agouti-related protein antagonist.

The melanocortin-4 receptor (MC4R) is a G-protein-coupled receptor (GPCR) that is expressed in the central nervous system and has a role in regulating feeding behavior, obesity, energy homeostasis, male erectile response, and blood pressure. Since the report of the MC4R knockout mouse in 1997, the field has been searching for links between this genetic biomarker and human obesity and type 2 diabetes. More then 80 single nucleotide polymorphisms (SNPs) have been identified from human patients, both obese and nonobese controls. Many significant studies have been performed examining the pharmacological characteristics of these hMC4R SNPs in attempts to identify a molecular defects/insights that might link a genetic factor to the obese phenotype observed in patients possessing these mutations. Our laboratory has previously reported the pharmacological characterization of 40 of these polymorphic hMC4 receptors with multiple endogenous and synthetic ligands. The goal of the current study is to perform a similar comprehensive side-by-side characterization of 30 additional human hMC4R with single nucleotide polymorphisms using multiple endogenous agonists [alpha-, beta-, and gamma(2)-melanocyte stimulating hormones (MSH) and adrenocorticotropin (ACTH)], the antagonist agouti-related protein hAGRP(87-132), and synthetic agonists [NDP-MSH, MTII, and the tetrapeptide Ac-His-dPhe-Arg-Trp-NH(2) (JRH887-9)]. These in vitro data, in some cases, provide a putative molecular link between dysfunctional hMC4R's and human obesity. These 30 hMC4R SNPs include R7H, R18H, R18L, S36Y, P48S, V50M, F51L, E61K, I69T, D90N, S94R, G98R, I121T, A154D, Y157S, W174C, G181D, F202L, A219 V, I226T, G231S, G238D, N240S, C271R, S295P, P299L, E308K, I317V, L325F, and 750DelGA. All but the N240S hMC4R were identified in obese patients. Additionally, we have characterized a double I102T/V103I hMC4R. In addition to the pharmacological characterization, the hMC4R variants were evaluated for cell surface expression by flow cytometry. The F51L, I69T, and A219V hMC4Rs possessed full agonist activity and significantly decreased endogenous agonist ligand potency. At the E61K, D90N, Y157S, and C271R hMC4Rs, all agonist ligands examined were only partially efficacious in generating a maximal signaling response (partial agonists) and possessed significantly decreased endogenous agonist ligand potency. Only the A219V, G238D, and S295P hMC4Rs possessed significantly decreased AGRP(87-132) antagonist potency. These data provide new information for use in GPCR computational development as well as insights into MC4R structure ad function.

Peptide and small molecules rescue the functional activity and agonist potency of dysfunctional human melanocortin-4 receptor polymorphisms.

The melanocortin pathway, specifically the melanocortin-4 receptor and the cognate endogenous agonist and antagonist ligands, have been strongly implicated in the regulation of energy homeostasis and satiety. Genetic studies of morbidly obese human patients and normal weight control patients have resulted in the discovery of over 70 human melanocortin-4 receptor (MC4R) polymorphisms observed as both heterozygous and homozygous forms. A number of laboratories have been studying these hMC4R polymorphisms attempting to understand the molecular mechanism(s) that might explain the obese human phenotype. Herein, we have studied 13 polymorphic hMC4Rs that have been identified to possess statistically significant decreased endogenous agonist potency with synthetic peptides and small molecules attempting to identify ligands that can pharmacologically rescue the hMC4R polymorphic agonist response. The ligands examined in this study include NDP-MSH, MTII, Ac-His-DPhe-Arg-Trp-NH2 (JRH887-9), Ac-Anc-DPhe-Arg-Trp-NH2 (amino-2-naphtylcarboxylic acid, Anc, JRH420-12), Ac-His-(pI)DPhe-Arg-Trp-NH2 (JRH322-18), chimeric AGRP-melanocortin based ligands (Tyr-c[Cys-His-DPhe-Arg-Trp-Asn-Ala-Phe-Cys]-Tyr-NH2, AMW3-130 and Ac-mini-(His-DPhe-Arg-Trp)-hAGRP-NH2, AMW3-106), and the small molecules JB25 and THIQ. The hMC4R polymorphisms included in this study are S58C, N97D, I102S, L106P, S127L, T150I, R165Q, R165W, L250Q, G252S, C271Y, Y287Stop, and I301T. These studies resulted in the NDP-MSH, MTII, AMW3-130, THIQ, and AMW3-106 ligands possessing nanomolar to subnanomolar agonist potency at the hMC4R polymorphisms examined in this study. Thus, these ligands could generically rescue the potency and stimulatory response of the abnormally functioning hMC4Rs studied and may provide tools to further clarify the molecular mechanism(s) involving these receptor modifications.

Upregulation of Foxp3 expression in mouse and human Treg is IL-2/STAT5 dependent: implications for the NOD STAT5B mutation in diabetes pathogenesis.

Regulatory T cells (Treg), characterized as CD4(+)/CD25(+hi) T cells, are critical for sustaining and promoting immune tolerance. Treg are highly dependent on IL-2 and IL-2 signaling to maintain their numbers and function and interruption of this pathway promotes autoimmunity. The transcription factor, Foxp3, is also required for Treg function as defective Foxp3 promotes autoimmunity in both mice and humans. We previously reported a point mutation in the DNA-binding domain of the NOD STAT5B gene that limits DNA binding when compared to wild-type STAT5 mice. Based on the presence of five STAT5B consensus sequences in the Foxp3 promotor, we hypothesized a critical linkage between IL-2 signaling/STAT5B and Foxp3 expression in Treg. Our data show IL-2 activates long-form (LF) STAT5 and sustains Foxp3 expression in Treg. In contrast, CD4(+)/CD25(-) T cells do not active LF STAT5 and do not express Foxp3 under the same conditions. In addition, blocking LF STAT5 activation with a Jak inhibitor (AG-490) significantly reduced Foxp3 expression in Treg. Examination of human Treg using flow cytometry and intracellular staining for Foxp3 expression likewise demonstrates that IL-2 maintains Foxp3 expression through LF STAT5 signaling. These studies reveal a critical link between IL-2 mediated JAK-STAT5 signaling and the maintenance of Foxp3 expression in Treg of mice and humans.

Pharmacological characterization of 40 human melanocortin-4 receptor polymorphisms with the endogenous proopiomelanocortin-derived agonists and the agouti-related protein (AGRP) antagonist.

The melanocortin-4 receptor (MC4R) is a G-protein coupled receptor (GPCR) that is expressed in the central nervous system and has a role in regulating energy homeostasis and obesity. Up to a remarkable 6% of morbidly obese adults and children studied possess single nucleotide polymorphisms (SNPs) of the MC4R. Upon stimulation by agonist, the MC4R signals through the intracellular adenylate cyclase signal transduction pathway. Posttranslational modification of the pro-opiomelanocortin (POMC) gene transcript results in the generation of several endogenous melanocortin receptor agonists including alpha-, beta-, gamma-melanocyte stimulating hormones (MSH) and adrenocorticotropin (ACTH) ligands. The endogenous MC4R antagonist, agouti-related protein (AGRP), is expressed in the brain and is only one of two naturally occurring antagonists of GPCRs identified to date. Herein, we have generated 40 hMC4 polymorphic receptors and evaluated their cell surface expression by flow cytometry as well as pharmacologically characterized their functionality using the endogenous agonists alpha-MSH, beta-MSH, gamma2-MSH, ACTH(1-24), the antagonist hAGRP(87-132), and the synthetic agonists NDP-MSH and MTII. This is the first study in which polymorphic hMC4Rs have been pharmacologically characterized simultaneously with multiple endogenous ligands. Interestingly, at the N97D, L106P, and C271Y hMC4Rs beta-MSH was more potent than the other endogenous agonists alpha-MSH, gamma2-MSH, ACTH(1-24). The S58C and R165Q/W hMC4Rs possessed significantly reduced endogenous agonist potency (15- to 90-fold), but the synthetic ligands NDP-MSH and MTII possessed only 2-9-fold reduced potency as compared to the wild-type receptor, suggesting their potential as therapeutic ligands to treat individuals with these polymorphisms.

Molecular mechanism of the constitutive activation of the L250Q human melanocortin-4 receptor polymorphism.

The Melanocortin-4 Receptor is a G-protein coupled receptor that has been physiologically linked to participate in the regulation of energy homeostasis. The Melanocortin-4 Receptor is stimulated by endogenous melanocortin agonists derived from the pro-opiomelanocortin gene transcript and antagonized by the endogenous antagonist agouti-related protein. Central administration of melanocortin agonists has been demonstrated to decrease food intake and conversely, treatment with antagonists resulted in increased food intake. Deletion of the Melanocortin-4 Receptor gene from the mouse genome results in an obese and hyperphagic phenotype. Polymorphisms of the human Melanocortin-4-Receptor have been found in severely obese individuals, suggesting that Melanocortin-4 Receptor malfunction might be involved in human obesity and obesity-associated diabetes. Herein, we have performed experiments to understand the molecular mechanisms associated with the L250Q human Melanocortin-4-Receptor polymorphism discovered in an extremely obese woman. This L250Q human Melanocortin-4-Receptor has been pharmacologically characterized to result in a constitutively active receptor. The fact that a constitutively active human Melanocortin-4-Receptor mutation was found in an obese person is a physiologic contradiction, as chronic activation of the human Melanocortin-4-Receptor and subsequently high cyclic adenosine monophosphate levels should theoretically result in a normal or lean phenotype. In this study, we demonstrated that agouti-related protein acts as an inverse agonist at this constitutively active receptor, and we propose a mechanism by which agouti-related protein might contribute to the obese phenotype in the L250Q patient. In addition, using receptor mutagenesis, pharmacology, and computer modeling approaches, we investigated the molecular mechanism by which modification of the L250 residue results in constitutive activation of the human Melanocortin-4-Receptor.