Mixed Lineage Kinase-3 Stabilizes and Functionally Cooperates with TRIBBLES-3 to Compromise Mitochondrial Integrity in Cytokine-induced Death of Pancreatic Beta Cells

Abstract

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 nonobese 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.In type 1 diabetes, the autoimmune destruction of pancreatic beta cells is driven by leukocyte infiltration and the damaging effects of locally secreted cytokines. Cytokines activate MAPKs 3 JNK and p38, via signaling modules that involve the sequential activation of a MAP3K, MAP2K, and MAPK, all scaffolded by a single protein (1). The existence of several families of MAP3Ks raises the possibility that each MAP3K may be activated by specific classes of stimuli. The serine-threonine MAP3K mixed lineage kinase-3 (MLK3) is activated by cytokines (2, 3) and assembles a signaling module consisting of MKK7, JNK, and the scaffold protein JIP1 (4, 5). Fibroblasts with a targeted deletion of either MKK7 or MLK3 are attenuated in their response to cytokines (6, 7). Elevation of MLK3 has been linked to induction of apoptosis in neurons (8 -10), and inhibition of MLKs can delay progression of neurodegenerative diseases (reviewed in Ref. 11 and studies quoted therein). The striking parallels between the beta cell and neuronal phenotypes, coupled with the ability of cytokines to activate MLK3, prompted us to examine whether MLKs participate in cytokine-induced beta cell death.Here we show that MLK3 is markedly elevated in leukocyteinfiltrated islets of the non-obese type 1 diabetic (NOD) mouse. To investigate the potential role of MLK3 in beta cell death, we devised an ex vivo system for co-culture of primary islets with immune-activated splenocytes. Compared with static culture with purified cytokines, this system is likely to be more representative of the milieu encountered by islets in autoimmune diabetes. We observed rapid activation of MLK3, and MLK3 was required for cytokine-mediated apoptosis via BAX, a proapoptotic member of the BCL-2 protein family. MLK3 mediated its effects via the pseudokinase TRB3 (TRIBBLES homolog 3), originally identified as an inducible factor in neuronal cell death (12) and subsequently shown to be a potent negative regulator of the prosurvival kinase Akt (13). We fo...

Prolonged insulin treatment sensitizes apoptosis pathways in pancreatic β cells

“…Insulin was also reported to induce apoptosis in glucosedeprived β cells (Guillen et al 2008) and potentiate the deleterious effects of H 2 O 2 on β cell survival (Sampson et al 2010). Of special interest in this regard is our finding that insulin increased the expression of iNos and Trb3, factors whose expression is known to be increased by cytokines in pancreatic β cells (Kanki et al 2009, Humphrey et al 2010, Kanwar 2010, Quintana-Lopez et al 2013. Trb3 was recently shown to mediate FFA-induced apoptosis in pancreatic β cells (Qin et al 2014).…”

“…Among the candidate ER stress proteins that might be involved in insulin effects is Trb3, a downstream target of Atf4, which has been found to be increased by cytokines (Kanki et al 2009, Humphrey et al 2010, Kanwar 2010),…”

Developmental roles of tribbles protein family members

“…In the N-terminus, mutations fail to induce AML in a Hox9 coexpression assay, while mutations in the COP1 binding site bind but fail to degrade C/EBP and also compromise the leukemogenic activity of Trib1 and Trib2. The Trib MEK1 motif ILLHPWF is thought to mediate interactions with multiple MAPkinase kinases (MAPKKs), including (1) Trib3 binding MLK3 in pancreatic b-cells (Humphrey et al, 2010); (2) Trib2 binding and inactivation of MKK7 and MEK1, involved in Erk and Jnk signaling, respectively (Eder et al, 2008); and (3) Trib1 binding and enhancing phosphorylation of ERK1/2, important for its leukemogenic activity (Yokoyama et al, 2010). Interdependence of these domains is suggested by Trib1 mutants that lack the MEK1 binding motif and fail to degrade C/EBPa, suggesting the COP1 function of Tribs is dependent on activation of the MEK1/ERK pathway .…”

“…While knockdown suppresses the effect of GMCSF withdrawal, overexpression of Trib2 stimulates apoptosis by means of a caspase, not the proteosome (Lin et al, 2007). Trib3 also promotes apoptosis in macrophages (Shang et al, 2009), gliomas (Salazar et al, 2009), pancreatic b-cells (Humphrey et al, 2010), and chondrocytes (Cravero et al, 2009). …”

Loss of TRB3 Alters Dynamics of MLK3-JNK Signaling and Inhibits Cytokine-activated Pancreatic Beta Cell Death

“…JNK activation has been clearly linked to mitochondrial stress and damage in multiple cell types, including the pancreatic beta cell (4 -6). We have reported that cytokine-induced MLK3-JNK pathway is instrumental in compromising mitochondrial integrity early in the apoptotic process, effectively reducing cellular defenses and increasing the potency of subsequent inflammatory events (6).…”