No abstract

The pancreatic ATP-sensitive potassium (K ATP ) channel, a complex of four sulfonylurea receptor 1 (SUR1) and four potassium channel Kir6.2 subunits, regulates insulin secretion by linking metabolic changes to ␤-cell membrane potential. Sulfonylureas inhibit K ATP channel activities by binding to SUR1 and are widely used to treat type II diabetes. We report here that sulfonylureas also function as chemical chaperones to rescue K ATP channel trafficking defects caused by two SUR1 mutations, A116P and V187D, identified in patients with congenital hyperinsulinism. Sulfonylureas markedly increased cell surface expression of the A116P and V187D mutants by stabilizing the mutant SUR1 proteins and promoting their maturation. By contrast, diazoxide, a potassium channel opener that also binds SUR1, had no effect on surface expression of either mutant. Importantly, both mutant channels rescued to the cell surface have normal ATP, MgADP, and diazoxide sensitivities, demonstrating that SUR1 harboring either the A116P or the V187D mutation is capable of associating with Kir6.2 to form functional K ATP channels. Thus, sulfonylureas may be used to treat congenital hyperinsulinism caused by certain K ATP channel trafficking mutations. ATP-sensitive potassium (K ATP )1 channels present in the plasma membrane of pancreatic ␤-cells play a central role in mediating glucose-induced insulin secretion (1-4). The activity of K ATP channels, which regulates ␤-cell membrane potential, is determined by the relative concentrations of intracellular ATP and ADP. When the blood glucose level rises, the increased intracellular [ATP/ADP] ratio favors K ATP channel closure, resulting in membrane depolarization, Ca 2ϩ influx, and insulin secretion. When the blood glucose level falls, the above molecular events reverse, and insulin release is stopped. In the event where K ATP channels fail to open during glucose starvation, ␤-cell membrane potential remains depolarized, and insulin secretion persists, leading to severe hypoglycemia. These symptoms are found in patients suffering from congenital hyperinsulinism (5), also known as persistent hyperinsulinemia hypoglycemia of infancy (PHHI) (6). Indeed, mutations in the K ATP channel genes, sulfonylurea receptor 1 (SUR1) and the inward rectifier potassium channel Kir6.2, that lead to a loss of channel function have been shown to be major causes of PHHI (4, 6).The pancreatic K ATP channel complex consists of four poreforming Kir6.2 subunits and four regulatory SUR1 subunits (7-10). Gating of K ATP channels occurs as a result of the interplay between both channel subunits and intracellular ATP and ADP. Binding of ATP to the Kir6.2 subunit inhibits channel activity, whereas binding of Mg 2ϩ -complexed ATP or ADP to the SUR1 subunit stimulates channel activity (11)(12)(13)(14). SUR1 is a member of the ATP-binding cassette transporter family; it has three transmembrane domains: TM0, TM1, and TM2, and two large cytoplasmic nucleotide binding domains: NBD1 and NBD2 (15,16). Structure-function studies sugges...

Histone deacetylase (HDAC) inhibition modulates dendritic cells (DCs) functions and regulates experimental graft-versus-host disease (GVHD) and other immune mediated diseases. The mechanisms by which HDAC inhibition modulates immune responses remain largely unknown. Signal transducer and activator of transcription-3 (STAT-3) is a transcription factor shown to negatively regulate DC functions. Herein we report that HDAC inhibition acetylates and activates STAT-3, which regulates DCs by promoting the transcription of indoleamine 2, 3-dioxygenase (IDO). These findings demonstrate (a) novel functional role for post-translational modification of STAT-3 through acetylation and (b) provide mechanistic insights into HDAC inhibition mediated immuno-regulation by induction of IDO.

Administration of exogenous interleukin-18 (IL-18) regulates experimental acute graftversus-host disease (GVHD) in a

Sema4D (CD100), a member of the neuro-semaphorin family of proteins, has recently been shown to play a role in modulating certain immune responses. We tested the requirement of Sema4D expression on T cells in the induction of T cell allo-immune responses. Sema4D-/- T cells showed reduced expansion in vitro upon stimulation with allo-geneic antigen presenting cells (APCs) when compared to wild-type (wt) T cells. Similar in vitro results were observed using anti-Sema4D mAbs. Further studies demonstrated that the reduced proliferation was not due to intrinsic T cell defects, and that the cytotoxic functions were preserved. After allo-geneic bone marrow transplant (BMT), recipients of Sema4D-/- T cells showed reduced mortality and graft-versus-host disease (GVHD) target organ damage. Allo-geneic dendritic cells (DCs) cocultured with Sema4D-/- responder T cells secreted less TNF-alpha and IL-12p70 compared to wt T cells. Similar reduction of DC function was observed with anti-Sema4D mAbs. Given the preservation of CTL function we evaluated graft-versus-leukemia (GVL) responses. When BALB/c recipient mice were challenged with the P815 murine mastocytoma cell line (H2(d)) the recipients of allo-geneic Sema4D-/- B6 T cells showed a significant improvement in tumor free survival when compared to syngeneic recipients, thus demonstrating preservation of GVL, albeit of a lesser magnitude than allo-geneic wt T cells. In summary, Sema4D plays a significant role in mediating in vitro and in vivo allo-geneic responses by modulating T cell-APC interactions.

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Histone deacetylase (HDAC) inhibitors reduce experimental acute graft-versus-host disease (GVHD) and recent work by us and others suggest that HDAC inhibitors regulate dendritic cell (DC) function. However, the critical cellular and molecular mechanisms underpinning these observations are not known. We investigated the mechanisms by utilizing two HDAC inhibitors, suberoylanilide hydroxamic acid (SAHA) and ITF 2357. Pretreatment of murine bone marrow (BM) and human peripheral blood mononuclear cell derived DCs with either HDAC inhibtors and stimulated with TLR ligands such as LPS caused a significant reduction in the secretion of TNF-α compared to the untreated controls (P< 0.01). Pre-treatment also significantly reduced the DC mediated in vitro and in vivo stimulation of allogeneic T cell proliferation (P<0.05). SAHA and ITF 2357 increased expression of indoleamine 2, 3-dioxygenase (IDO) at both mRNA and protein levels. Blockade of IDO induction with specific small interfering RNA (siRNA) in the wild type (WT) DCs and those derived from IDO deficient (IDO−/−) animals confirmed a functional role for IDO in the HDAC inhibitor mediated regulation TNFα secretion and allo-T cell proliferation. DNA-protein interaction analysis with ChIP assay demonstrated that both acetylated histone(H) 4 and STAT3 bound to murine IDO promoter. Using TESS DNA soft-wear analysis we found two potential STAT3 binding Gamma Activated Sites (GAS sites) in the IDO promoter and it was recently reported that acetylation of STAT3 is sufficient for its activation (Yuan, et al. Science 2005). We therefore sought to determine whether direct acetylation of STAT3 by the HDAC inhibitors is critical for the induction of IDO in DCs. SAHA or ITF2357 treatment induced acetylation, activation and dimerization of STAT-3 as determined by protein-protein interaction studies. Co-culture studies with JSI-124, an inhibitor of STAT3 signaling, demonstrated that STAT3 is critical for induction of IDO by the HDAC inhibitors. Functional relevance was confirmed by the lack of HDAC inhibitor induced suppression of DC function when co-treated with JSI-124. We next cloned 1500bp DNA fragment upstream of mouse IDO gene start codon and attached it to luciferase gene and peformed mutagenesis studies to evaluate for luciferase activity. Deletion of the GAS regions or treatment with JSI-124 impaired luciferase activity of the IDO promoter constructs demonstrating that STAT3 is necessary and sufficient for transcriptional induction of IDO by the HDAC inhibitors. To specifically address the in vivo relevance of IDO induction by HDAC inhibitors in only the host type DCs, we generated [B6 → B6] and [IDO−/−B6 → B6] BM chimeras and utilized them in a well characterized [BALB/c B6] mouse model of acute GVHD. [B6 → B6] and [IDO−/− B6 → B6] animals received 800 Gy on day −1 and were treated orally with of 50mg/kg of ITF 2357 or diluent on days −1 to +2. Mice were transplanted on day 0 with 3 x 106 T cells and 5 x 106 BM from either syngeneic B6 or allogeneic BALB/c donors. Treatment with ITF 2357 resulted in significantly better survival in the allogeneic [B6 → B6] animals (80% vs. 40%, P < 0.02) but did not confer any survival benefit to the [IDO−/− B6 → B6] animals when compared with diluent treated recipients [20% vs. 30%, P = NS]. Our data thus demonstrate a novel molecular pathway in modulation of GVHD through a STAT3 dependent induction of IDO in the host DCs.