First person – Hannah Black and Rachel Livingstone

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Hannah Black and Rachel Livingstone are co-first authors on ‘ Knockout of syntaxin-4 in 3T3-L1 adipocytes reveals new insight into GLUT4 trafficking and adiponectin secretion’, published in JCS. Hannah conducted the research described in this article while a PhD student in Professor Nia Bryant and Professor Gwyn Gould's lab at the Henry Wellcome Laboratory for Cell Biology, University of Glasgow, UK. She is now a postdoc in the lab of Professor Nia Bryant at the Department of Biology, University of York, UK, investigating membrane trafficking of the glucose transporter protein GLUT4. Rachel is a PhD student in the lab of Professor Gwyn Gould at the Henry Wellcome Laboratory for Cell Biology, University of Glasgow, UK, where she is also investigating membrane trafficking of GLUT4.

Knockout of Syntaxin-4 in 3T3-L1 adipocytes reveals new insight into GLUT4 trafficking and adiponectin secretion

Adipocytes are key to metabolic regulation, exhibiting insulin-stimulated glucose transport which is underpinned by the insulin-stimulated delivery of glucose transporter-4 (GLUT4)- containing vesicles to the plasma membrane where they dock and fuse increasing cell surface GLUT4 levels. Adipocytokines such as adiponectin are secreted via a similar mechanism. We used genome editing to knockout Syntaxin-4 a protein reported to mediate GLUT4-vesicle fusion with the plasma membrane in 3T3-L1 adipocytes. Syntaxin-4 knockout reduced insulin-stimulated glucose transport and adiponectin secretion by ∼50% and reduced GLUT4 levels. Ectopic expression of HA-GLUT4-GFP showed that Syntaxin-4 knockout cells retain significant GLUT4 translocation capacity demonstrating that Syntaxin-4 is dispensable for insulin-stimulated GLUT4 translocation. Analysis of recycling kinetics revealed only a modest reduction in the exocytic rate of GLUT4 in knockout cells, and little effect on endocytosis. These analyses demonstrate that Syntaxin-4 is not always rate limiting for GLUT4 delivery to the cell surface. In sum, we show that Syntaxin-4 knockout results in reduced insulin-stimulated glucose transport, depletion of cellular GLUT4 levels and inhibition of adiponectin secretion but has only modest effects on the translocation capacity of the cells.

Syntaxin 4 Eenrichment in β-cells Prevents Conversion to Autoimmune Diabetes in Non-Obese Diabetic (NOD) Mice

Syntaxin 4 (STX4), a plasma membrane-localized SNARE protein, regulates human islet β-cell insulin secretion and preservation of β-cell mass. We found that human type 1 diabetic (T1D) and non-obese diabetic (NOD) mouse islets show reduced β-cell STX4 expression, consistent with decreased STX4 expression as a potential driver of T1D phenotypes. To test this hypothesis, we generated inducible β-cell-specific STX4-expressing NOD mice (NOD-iβSTX4).<b> </b>Of NOD-iβSTX4 mice, 73% had sustained normoglycemia versus <20% of control NOD (NOD-Ctrl) mice, by 25 weeks of age. At 12 weeks of age, prior to diabetes conversion, NOD-iβSTX4 mice demonstrated superior whole-body glucose tolerance and β-cell glucose responsiveness than NOD-Ctrl mice. Higher β-cell mass and reduced β-cell apoptosis were also detected in NOD-iβSTX4 pancreata compared with those of NOD-Ctrl mice. Single-cell RNA‐sequencing revealed that islets from NOD-iβSTX4 had markedly reduced IFNƔ signaling and TNFα signaling via NF-ĸB in islet β-cells, including reduced expression of the chemokine CCL5; CD4⁺ Treg cells were also enriched in NOD-iβSTX4 islets. These results provide a deeper mechanistic understanding of STX4 function in β-cell protection and warrant further investigation of STX4 enrichment as a strategy to reverse or prevent T1D in humans or protect β-cell grafts.

Syntaxin 4 Eenrichment in β-cells Prevents Conversion to Autoimmune Diabetes in Non-Obese Diabetic (NOD) Mice

Syntaxin 4 (STX4), a plasma membrane-localized SNARE protein, regulates human islet β-cell insulin secretion and preservation of β-cell mass. We found that human type 1 diabetic (T1D) and non-obese diabetic (NOD) mouse islets show reduced β-cell STX4 expression, consistent with decreased STX4 expression as a potential driver of T1D phenotypes. To test this hypothesis, we generated inducible β-cell-specific STX4-expressing NOD mice (NOD-iβSTX4).<b> </b>Of NOD-iβSTX4 mice, 73% had sustained normoglycemia versus <20% of control NOD (NOD-Ctrl) mice, by 25 weeks of age. At 12 weeks of age, prior to diabetes conversion, NOD-iβSTX4 mice demonstrated superior whole-body glucose tolerance and β-cell glucose responsiveness than NOD-Ctrl mice. Higher β-cell mass and reduced β-cell apoptosis were also detected in NOD-iβSTX4 pancreata compared with those of NOD-Ctrl mice. Single-cell RNA‐sequencing revealed that islets from NOD-iβSTX4 had markedly reduced IFNƔ signaling and TNFα signaling via NF-ĸB in islet β-cells, including reduced expression of the chemokine CCL5; CD4⁺ Treg cells were also enriched in NOD-iβSTX4 islets. These results provide a deeper mechanistic understanding of STX4 function in β-cell protection and warrant further investigation of STX4 enrichment as a strategy to reverse or prevent T1D in humans or protect β-cell grafts.

Syntaxin 4 Mediates NF-κB Signaling and Chemokine Ligand Expression via Specific Interaction with IκBβ

Enrichment of human islets with Syntaxin 4 (STX4) improves functional β-cell mass through a nuclear factor- kB (NF-kB)-dependent mechanism. However, the detailed mechanisms underlying the protective effect of STX4 are unknown. To determine the signaling events linking STX4 enrichment and downregulation of NF-kB activity, STX4 was overexpressed in human islets, EndoC-βH1 and INS-1 832/13 cells in culture, and the cells were challenged with the proinflammatory cytokines interleukin-1β, tumor necrosis factor-a and interferon-g, individually and in combination. STX4 expression suppressed cytokine-induced proteasomal degradation of IkBβ but not IkBa. Inhibition of IKKβ prevented IkBβ degradation, suggesting that IKKβ phosphorylates IkBβ. Moreover, the IKKβ inhibitor, as well as a proteosomal degradation inhibitor, prevented the loss of STX4 caused by cytokines. This suggests that STX4 may be phosphorylated by IKKβ in response to cytokines, targeting STX4 for proteosomal degradation. Expression of a stabilized form of STX4 further protected IkBβ from proteasomal degradation, and like wildtype STX4, stabilized STX4 coimmunoprecipitated with IkBβ and the NF-kB p50 subunit. This work proposes a novel pathway wherein STX4 regulates cytokine-induced NF-kB signaling in β-cells <i>via</i> associating with and preventing IkBβ degradation, suppressing chemokine expression, and protecting islet β-cells from cytokine-mediated dysfunction and demise.

Syntaxin 4 Mediates NF-κB Signaling and Chemokine Ligand Expression via Specific Interaction with IκBβ

Enrichment of human islets with Syntaxin 4 (STX4) improves functional β-cell mass through a nuclear factor- kB (NF-kB)-dependent mechanism. However, the detailed mechanisms underlying the protective effect of STX4 are unknown. To determine the signaling events linking STX4 enrichment and downregulation of NF-kB activity, STX4 was overexpressed in human islets, EndoC-βH1 and INS-1 832/13 cells in culture, and the cells were challenged with the proinflammatory cytokines interleukin-1β, tumor necrosis factor-a and interferon-g, individually and in combination. STX4 expression suppressed cytokine-induced proteasomal degradation of IkBβ but not IkBa. Inhibition of IKKβ prevented IkBβ degradation, suggesting that IKKβ phosphorylates IkBβ. Moreover, the IKKβ inhibitor, as well as a proteosomal degradation inhibitor, prevented the loss of STX4 caused by cytokines. This suggests that STX4 may be phosphorylated by IKKβ in response to cytokines, targeting STX4 for proteosomal degradation. Expression of a stabilized form of STX4 further protected IkBβ from proteasomal degradation, and like wildtype STX4, stabilized STX4 coimmunoprecipitated with IkBβ and the NF-kB p50 subunit. This work proposes a novel pathway wherein STX4 regulates cytokine-induced NF-kB signaling in β-cells <i>via</i> associating with and preventing IkBβ degradation, suppressing chemokine expression, and protecting islet β-cells from cytokine-mediated dysfunction and demise.

ELKS1 controls mast cell degranulation by regulating the transcription of Stxbp2 and Syntaxin 4 via Kdm2b stabilization

ELKS1 is a protein with proposed roles in regulated exocytosis in neurons and nuclear factor κB (NF-κB) signaling in cancer cells. However, how these two potential roles come together under physiological settings remain unknown. Since both regulated exocytosis and NF-κB signaling are determinants of mast cell (MC) functions, we generated mice lacking ELKS1 in connective tissue MCs (Elks1f/f Mcpt5-Cre) and found that while ELKS1 is dispensable for NF-κB–mediated cytokine production, it is essential for MC degranulation both in vivo and in vitro. Impaired degranulation was caused by reduced transcription of Syntaxin 4 (STX4) and Syntaxin binding protein 2 (Stxpb2), resulting from a lack of ELKS1-mediated stabilization of lysine-specific demethylase 2B (Kdm2b), which is an essential regulator of STX4 and Stxbp2 transcription. These results suggest a transcriptional role for active-zone proteins like ELKS1 and suggest that they may regulate exocytosis through a novel mechanism involving transcription of key exocytosis proteins.