Stress-induced pseudokinase TRB3 augments IL1ß signaling by interacting with Flightless homolog 1.

Interleukin-1ß is one of the most potent inducers of beta cell inflammation in the lead-up to type 1 diabetes. We have previously reported that IL1ß-stimulated pancreatic islets from mice with genetic ablation of stress-induced pseudokinase TRB3(TRB3KO) show attenuated activation kinetics for the MAP3K MLK3 and JNK stress kinases. However, JNK signaling constitutes only a portion of the cytokine-induced inflammatory response. Here we report that TRB3KO islets also show a decrease in amplitude and duration of IL1ß-induced phosphorylation of TAK1 and IKK, kinases that drive the potent NF-κB proinflammatory signaling pathway. We observed that TRB3KO islets display decreased cytokine-induced beta cell death, preceded by a decrease in select downstream NF-κB targets, including iNOS/NOS2 (inducible nitric oxide synthase), a mediator of beta cell dysfunction and death. Thus, loss of TRB3 attenuates both pathways required for a cytokine-inducible, proapoptotic response in beta cells. In order to better understand the molecular basis of TRB3-enhanced, post-receptor IL1ß signaling, we interrogated the TRB3 interactome using coimmunoprecipitation followed by mass spectrometry to identify immunomodulatory protein Flightless homolog 1 (Fli1) as a novel, TRB3-interacting protein. We show that TRB3 binds and disrupts Fli1-dependent sequestration of MyD88, thereby increasing availability of this most proximal adaptor required for IL1ß receptor-dependent signaling. Fli1 sequesters MyD88 in a multiprotein complex resulting in a brake on the assembly of downstream signaling complexes. By interacting with Fli1, we propose that TRB3 lifts the brake on IL1ß signaling to augment the proinflammatory response in beta cells.

Loss of TRB3 alters dynamics of MLK3-JNK signaling and inhibits cytokine-activated pancreatic beta cell death.

Disabling cellular defense mechanisms is essential for induction of apoptosis. We have previously shown that cytokine-mediated activation of the MAP3K MLK3 stabilizes TRB3 protein levels to inhibit AKT and compromise beta cell survival. Here, we show that genetic deletion of TRB3 results in basal activation of AKT, preserves mitochondrial integrity, and confers resistance against cytokine-induced pancreatic beta cell death. Mechanistically, we find that TRB3 stabilizes MLK3, most likely by suppressing AKT-directed phosphorylation, ubiquitination, and proteasomal degradation of MLK3. Accordingly, TRB3(-/-) islets show a decrease in both the amplitude and duration of cytokine-stimulated MLK3 induction and JNK activation. It is well known that JNK signaling is facilitated by a feed forward loop of sequential kinase phosphorylation and is reinforced by a mutual stabilization of the module components. The failure of TRB3(-/-) islets to mount an optimal JNK activation response, coupled with the ability of TRB3 to engage and maintain steady state levels of MLK3, recasts TRB3 as an integral functional component of the JNK module in pancreatic beta cells.

Specification of hepatopancreas progenitors in zebrafish by hnf1ba and wnt2bb.

Although the liver and ventral pancreas are thought to arise from a common multipotent progenitor pool, it is unclear whether these progenitors of the hepatopancreas system are specified by a common genetic mechanism. Efforts to determine the role of Hnf1b and Wnt signaling in this crucial process have been confounded by a combination of factors, including a narrow time frame for hepatopancreas specification, functional redundancy among Wnt ligands, and pleiotropic defects caused by either severe loss of Wnt signaling or Hnf1b function. Using a novel hypomorphic hnf1ba zebrafish mutant that exhibits pancreas hypoplasia, as observed in HNF1B monogenic diabetes, we show that hnf1ba plays essential roles in regulating ß-cell number and pancreas specification, distinct from its function in regulating pancreas size and liver specification, respectively. By combining Hnf1ba partial loss of function with conditional loss of Wnt signaling, we uncover a crucial developmental window when these pathways synergize to specify the entire ventrally derived hepatopancreas progenitor population. Furthermore, our in vivo genetic studies demonstrate that hnf1ba generates a permissive domain for Wnt signaling activity in the foregut endoderm. Collectively, our findings provide a new model for HNF1B function, yield insight into pancreas and ß-cell development, and suggest a new mechanism for hepatopancreatic specification.

Lysine 63-linked ubiquitination modulates mixed lineage kinase-3 interaction with JIP1 scaffold protein in cytokine-induced pancreatic ß cell death.

The mixed lineage kinase MLK3 plays a crucial role in compromising mitochondrial integrity and functions as a proapoptotic competence factor in the early stages of cytokine-induced pancreatic ß cell death. In an effort to identify mechanisms that regulate MLK3 activity in ß cells, we discovered that IL-1ß stimulates Lys-63-linked ubiquitination of MLK3 via a conserved, TRAF6-binding peptapeptide motif in the catalytic domain of the kinase. TRAF6-mediated ubiquitination was required for dissociation of inactive monomeric MLK3 from the scaffold protein IB1/JIP1, facilitating the subsequent dimerization, autophosphorylation, and catalytic activation of MLK3. Inability to ubiquitinate MLK3, or the presence of A20, an upstream Lys-63-linked deubiquitinase, strongly curtailed the ability of MLK3 to affect the proapoptotic translocation of BAX in cytokine-stimulated pancreatic ß cells, an early step in the progression toward ß cell death. These studies suggest a novel mechanism for MLK3 activation and provide new clues for therapeutic intervention in promoting ß cell survival.

Mixed lineage kinase-3 stabilizes and functionally cooperates with TRIBBLES-3 to compromise mitochondrial integrity in cytokine-induced death of pancreatic beta cells.

Mixed lineage kinases (MLKs) have been implicated in cytokine signaling as well as in cell death pathways. Our studies show that MLK3 is activated in leukocyte-infiltrated islets of non-obese diabetic mice and that MLK3 activation compromises mitochondrial integrity and induces apoptosis of beta cells. Using an ex vivo model of islet-splenocyte co-culture, we show that MLK3 mediates its effects via the pseudokinase TRB3, a mammalian homolog of Drosophila Tribbles. TRB3 expression strongly coincided with conformational change and mitochondrial translocation of BAX. Mechanistically, MLK3 directly interacted with and stabilized TRB3, resulting in inhibition of Akt, a strong suppressor of BAX translocation and mitochondrial membrane permeabilization. Accordingly, attenuation of MLK3 or TRB3 expression each prevented cytokine-induced BAX conformational change and attenuated the progression to apoptosis. We conclude that MLKs compromise mitochondrial integrity and suppress cellular survival mechanisms via TRB3-dependent inhibition of Akt.

Glucose regulates steady-state levels of PDX1 via the reciprocal actions of GSK3 and AKT kinases.

The pancreatic beta cell is sensitive to even small changes in PDX1 protein levels; consequently, Pdx1 haploinsufficiency can inhibit beta cell growth and decrease insulin biosynthesis and gene expression, leading to compromised glucose-stimulated insulin secretion. Using metabolic labeling of primary islets and a cultured beta cell line, we show that glucose levels modulate PDX1 protein phosphorylation at a novel C-terminal GSK3 consensus that maps to serines 268 and 272. A decrease in glucose levels triggers increased turnover of the PDX1 protein in a GSK3-dependent manner, such that PDX1 phosphomutants are refractory to the destabilizing effect of low glucose. Glucose-stimulated activation of AKT and inhibition of GSK3 decrease PDX1 phosphorylation and delay degradation. Furthermore, direct pharmacologic inhibition of AKT destabilizes, and inhibition of GSK3 increases PDX1 protein stability. These studies define a novel functional role for the PDX1 C terminus in mediating the effects of glucose and demonstrate that glucose modulates PDX1 stability via the AKT-GSK3 axis.

Maintenance of pluripotency in human embryonic stem cells is STAT3 independent.

The preservation of "stemness" in mouse embryonic stem (mES) cells is maintained through a signal transduction pathway that requires the gp130 receptor, the interleukin-6 (IL-6) family of cytokines, and the Janus Kinase-signal transducer and activator (JAK/STAT) pathway. The factors and signaling pathways that regulate "stemness" in human embryonic stem (hES) cells remain to be elucidated. Here we report that STAT3 activation is not sufficient to block hES cell differentiation when the cells are grown on mouse feeder cells or when they are treated with conditioned media from feeder cells. Human ES cells differentiate in the presence of members of the IL-6 family of cytokines including leukemia inhibitory factor (LIF) and IL-6 or in the presence of the designer cytokine hyper-IL-6, which is a complex of soluble interleukin-6 receptor (IL-6R) and IL-6 with greatly enhanced bioactivity. Human ES cells express LIF, IL-6, and gp130 receptors, as well as the downstream signaling molecules. Stimulation of human and mouse ES cells with gp130 cytokines resulted in a robust phosphorylation of downstream ERK1, ERK2, and Akt kinases, as well as the STAT3 transcription factor. Loss of the pluripotency markers Nanog, Oct-4, and TRA-1-60 was observed in hES cells during gp130-dependent signaling, indicating that signaling through this pathway is insufficient to prevent the onset of differentiation. These data underscore a fundamental difference in requirements of murine versus hES cells. Furthermore, the data demonstrate the existence of an as-yet-unidentified factor in the conditioned media of mouse feeder layer cells that acts to maintain hES cell renewal in a STAT3-independent manner.

Characterization and isolation of promoter-defined nestin-positive cells from the human fetal pancreas.

Studies using adult human islets and mouse embryonic stem cells have suggested that the neurepithelial precursor cell marker nestin also identifies and can be used to purify beta-cell precursors. To determine whether nestin can be used to identify beta-cell progenitors in the developing human pancreas, we characterized nestin expression from 12 to 24 gestational weeks, purified nestin+ cells using an enhancer/promoter-driven selection plasmid, and determined whether nestin+ cells can differentiate into beta-cells. Nestin was visualized in the platelet endothelial cell adhesion molecule and alpha smooth muscle actin-positive blood vessels and colocalized with vimentin in the interstitium. Nestin was not observed in pan cytokeratin (pCK)-positive ductal epithelium or insulin cells. Purified nestin+ cells also coexpressed vimentin and lacked pCK immunoreactivity. Purified adult and fetal pancreatic fibroblasts also expressed nestin. The nestin enhancer/promoter used in the selection plasmid was sufficient to drive reporter gene expression, green fluorescent protein, in human fetal pancreatic tissue. Exposure of selected nestin+ cells to nicotinamide, hepatocyte growth factor/scatter factor, betacellulin, activin A, or exendin-4 failed to induce pancreatic and duodenal homeobox gene-1 or insulin message as determined by RT-PCR. Transplantation of nestin+ cells and fetal pancreatic fibroblasts into athymic mice also failed to result in the development of beta-cells, whereas nestin- fetal pancreatic epithelial cells gave rise to functional insulin-secreting beta-cells. We conclude that nestin is not a specific marker of beta-cell precursors in the developing human pancreas.

Regulation of pancreatic cell differentiation and morphogenesis.

Organogenesis requires tissue interactions to initiate the cascade of inductive and repressive signals necessary for normal organ development. Tissue interactions initiate the pancreatic lineage within the primitive foregut endodermal epithelium and continue to direct the morphogenesis and differentiation of the endocrine, exocrine and ductal portions of the pancreas. An understanding of the mechanisms controlling pancreatic growth would enable the development of alternative therapies for diseases such as type 1 diabetes.