Generation of the lipid messenger phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) is crucial for development, cell growth and survival, and motility, and it becomes dysfunctional in many diseases including cancers. Here we reveal a mechanism for PtdIns(3,4,5)P3 generation by scaffolded phosphoinositide kinases. In this pathway, class I phosphatidylinositol-3-OH kinase (PI(3)K) is assembled by IQGAP1 with PI(4)KIIIα and PIPKIα, which sequentially generate PtdIns(3,4,5)P3 from phosphatidylinositol. By scaffolding these kinases into functional proximity, the PtdIns(4,5)P2 generated is selectively used by PI(3)K for PtdIns(3,4,5)P3 generation, which then signals to PDK1 and Akt that are also in the complex. Moreover, multiple receptor types stimulate the assembly of this IQGAP1–PI(3)K signalling complex. Blockade of IQGAP1 interaction with PIPKIα or PI(3)K inhibited PtdIns(3,4,5)P3 generation and signalling, and selectively diminished cancer cell survival, revealing a target for cancer chemotherapy.
Toll-like receptor (TLR) 2 on antigen-presenting cells (APCs) enables these cells to recognize peptidoglycan-embedded lipopeptides and glycopolymers in the Staphylococcus aureus cell wall and mount an inflammatory response to this microbe. TLR2 signalling can also modulate immunity to S. aureus by inducing an interleukin (IL)-10 response in APCs. What determines the balance between proinflammatory and modulatory responses to S. aureus is unknown. We show that the modulatory IL-10 response preferentially occurs upon CD14- and CD36-independent TLR2 signaling, triggering PI3K activation, and is restricted to monocytes and monocyte-derived macrophages (MΦs). In contrast, monocyte-derived dendritic cells (DCs) produce mostly IL-12 and IL-23. The differential APC polarization induced by staphylococcal peptidoglycan translates into differential T helper responses: MΦs primarily trigger IL-10 and weak IL-17 responses, whereas DCs trigger a robust Th1/Th17 response. Exploitation of TLR2 signalling plasticity by S. aureus may explain the wide range of outcomes of human encounters with this microbe.
Pannexin 1 (PANX1) is a channel-forming glycoprotein expressed in many tissues including the skin. PANX1 channels allow the passage of ions and molecules up to 1 kDa, including ATP and other metabolites. In this study, we show that PANX1 is highly expressed in human melanoma tumors at all stages of disease progression, as well as in patient-derived cells and established melanoma cell lines. Reducing PANX1 protein levels using shRNA or inhibiting channel function with the channel blockers, carbenoxolone (CBX) and probenecid (PBN), significantly decreased cell growth and migration, and increased melanin production in A375-P and A375-MA2 cell lines. Further, treatment of A375-MA2 tumors in chicken embryo xenografts with CBX or PBN significantly reduced melanoma tumor weight and invasiveness. Blocking PANX1 channels with PBN reduced ATP release in A375-P cells, suggesting a potential role for PANX1 in purinergic signaling of melanoma cells. In addition, cell-surface biotinylation assays indicate that there is an intracellular pool of PANX1 in melanoma cells. PANX1 likely modulates signaling through the Wnt/β-catenin pathway, because β-catenin levels were significantly decreased upon PANX1 silencing. Collectively, our findings identify a role for PANX1 in controlling growth and tumorigenic properties of melanoma cells contributing to signaling pathways that modulate melanoma progression.
During development, the Hippo signaling pathway regulates key physiological processes, such as control of organ size, regeneration, and stem cell biology. Yes-associated protein (YAP) is a major transcriptional co-activator of the Hippo pathway. The scaffold protein IQGAP1 interacts with more than 100 binding partners to integrate diverse signaling pathways. In this study, we report that IQGAP1 binds to YAP and modulates its activity. The Hippo signaling pathway comprises numerous proteins that regulate organ size and shape, regeneration, and stem cell biology (1). The Hippo pathway responds to several stimuli, such as stress, polarity, and adhesion cues and has been implicated in tumorigenesis (2). The primary components of this cascade are a kinase module and a transcriptional module. The transcriptional module consists of yes-associated protein (YAP) 2 and the transcriptional co-activator with PDZ-binding motif (TAZ), which together drive the activities downstream of the Hippo pathway (3). Importantly, YAP shuttles between the cytoplasm and nucleus. Nevertheless, nuclear translocation of YAP is not sufficient for induction of transcriptional activity because YAP does not contain a DNA-binding region. Nuclear YAP activity is elicited by binding to transcription factors. Among these, TEA domain family members (TEADs) are the major transcription factors driving YAP-mediated gene transcription (4, 5). YAP activity is regulated through phosphorylation-dependent and -independent mechanisms. When Hippo is ON, YAP gets phosphorylated, and its co-transcriptional activity is inhibited. The kinase module of the Hippo pathway, including large tumor suppressor 1 and 2 (LATS1/2) and mammalian STE20-like protein kinase 1 and 2 (MST1/2), inhibits YAP by catalyzing phosphorylation at Ser 127 or Ser 381 (6). Phosphorylation of YAP at Ser 127 induces its interaction with 14-3-3, which leads to retention of YAP in the cytoplasm, thereby inhibiting transcriptional activity. Phosphorylation of YAP at Ser 381 (Ser 381 in mice corresponds to residue Ser 397 in humans; all reference to phosphorylation at this site will be described as Ser 381 ) leads to its ubiquitination and cytoplasmic degradation. Furthermore, other regulatory proteins, such as NF2 (neurofibromatin 2) and KIBRA (kidney and brain expressed protein), form a complex to activate the Hippo kinase module, thereby inhibiting YAP nuclear activity (7). YAP can also be modulated by direct protein-protein interactions. For example, the angiomotin (AMOT) family of proteins binds to YAP and promotes its cytoplasmic retention (8). In contrast, direct binding of YAP to multiple ankyrin repeats single KH domain-containing protein (MASK) in the nucleus potentiates YAP-mediated transcriptional activity (9).IQGAPs are evolutionary conserved, scaffold proteins with multiple functions. Three IQGAP family members (named IQGAP1, IQGAP2, and IQGAP3) have been identified in mammals (10). The expression of IQGAP2 is restricted to liver (11), and IQGAP3 is reported to be expressed in lung...
Pannexin 1 (Panx1) is a channel-forming glycoprotein important in paracrine signaling and cellular development. In this study, we discovered that mice globally lacking Panx1 (KO) have significantly greater total fat mass and reduced lean mass compared to wild type (WT) mice under a normal diet. Despite having higher fat content, Panx1 KO mice on a high fat diet exhibited no differences in weight gain and blood markers of obesity as compared to WT controls, except for an increase in glucose and insulin levels. However, metabolic cage data revealed that these Panx1 KO mice display significantly increased activity levels, higher ambulatory activity, and reduced sleep duration relative to their WT littermates on a high-fat diet. To uncover the cellular mechanism responsible for the increased fat content in the KO, we isolated primary cultures of adipose-derived stromal cells (ASCs) from WT and KO fat pads. In WT ASCs we observed that Panx1 protein levels increase upon induction into an adipogenic lineage. ASCs isolated from Panx1 KO mice proliferate less but demonstrate enhanced adipogenic differentiation with increased intracellular lipid accumulation, glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity, and adipokine secretion, as compared to WT ASCs. This was consistent with the increased adipocyte size and decreased adipocyte numbers observed in subcutaneous fat of the Panx1 KO mice compared to WT. We concluded that Panx1 plays a key role in adipose stromal cells during the early stages of adipogenic proliferation and differentiation, regulating fat accumulation in vivo.
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