School of Medicine

Mammalian α-arrestins are members of the same arrestin family as the ubiquitously expressed and extensively studied β-arrestins. Arrestins share common structural elements, including the conserved N- and C-arrestin-fold domains, polar core, finger loop, and C-terminal tail, all of which mediate protein-protein interactions. In β-arrestins, these domains enable the control of G protein-coupled receptor (GPCR) signaling and scaffolding interactions with various signaling proteins including c-Src. By contrast, the repertoire of α-arrestin scaffolding partners and regulatory mechanisms that control their interactions are not well-understood. α-arrestins differ considerably from β-arrestins in the C-terminal region; β-arrestins contain clathrin adaptor β-adaptin-binding sites, whereas α-arrestins harbor PPxY motifs, demonstrated to interact with WW domains of E3 ubiquitin ligases such as WWP2. Here we report the identification of a novel phosphorylation site, tyrosine (Y) 394, embedded in the C-terminal PPxY motif of α-arrestin ARRDC3. The Y394 site functions as a phospho-regulatory switch to enable distinct ARRDC3 binding partners and scaffolding functions. We found that ARRDC3 Y394 phosphorylation promotes interaction with c-Src via its SH2 domain, whereas the non-phosphorylated form binds to WWP2. Our results further show that ARRDC3 Y394 phosphorylation and c-Src SH2 domain-dependent interaction enables regulation of c-Src activity, whereas ARRDC3 Y394 phosphorylation disrupts WWP2 interaction and perturbs ARRDC3-dependent lysosomal trafficking of the GPCR, protease-activated receptor-1. Together, these findings indicate that ARRDC3 Y394 functions as a phospho-regulatory switch to enable selective binding to different partners that impact distinct scaffolding functions.

Monocytes infiltrating tumors acquire various states that distinctly impact cancer treatment. Here, we show that resistance of tumors to radiotherapy (RT) is controlled by the accumulation of monocyte-derived dendritic cells (moDCs). These moDCs are characterized by the expression of CD301b and have a superior capacity to generate regulatory T cells (Tregs). Accordingly, moDC depletion limits Treg generation and improves the therapeutic outcome of RT. Mechanistically, we demonstrate that granulocyte-macrophage colony-stimulating factor (GM-CSF) derived from radioresistant tumor cells following RT is necessary for the accumulation of moDCs. Our results unravel the immunosuppressive function of moDCs and identify GM-CSF as an immunotherapeutic target during RT.

Dual inhibition of sodium glucose cotransporters 1 and 2 (SGLT1/SGLT2) by sotagliflozin protects the kidney and heart in patients with type 2 diabetes mellitus (T2DM) and chronic kidney disease (CKD). To gain mechanistic insights, the current study aimed to establish a murine model of hypertensive CKD that shows cardio-renal protection by sotagliflozin. Since protection by SGLT2 inhibitors can be diabetes-independent, a nondiabetic murine model of subtotal nephrectomy with angiotensin II infusion-facilitated hypertension was followed for 7 weeks. The model showed 40% lower GFR, doubling in plasma FGF23, 50 mmHg higher systolic blood pressure (SBP), 100-fold increased albuminuria, and robust signs of kidney injury, inflammation, and fibrosis versus sham controls, associated with a 30% larger left cardiac ventricle and wall thickness and upregulation of markers of cardiac overload and fibrosis. Sotagliflozin, initiated 1 week after the last surgery, showed target-engagement evidenced by glucosuria, 9 mmHg lower SBP, temporal reduction in body weight and GFR, and 30% higher plasma GLP1. Sotagliflozin, however, did not improve markers of kidney injury, inflammation, fibrosis, albuminuria, and plasma FGF23, or signs of cardiac overload, fibrosis, or impaired function. Limited sotagliflozin responsiveness may relate to short treatment time, limited metabolic benefits in nondiabetic setting and/or the model's dominant angiotensin II-driven effects/hypertension.

Signaling by G protein-coupled receptors (GPCRs) is regulated by temporally distinct processes including receptor desensitization, internalization, and lysosomal sorting, and are tightly controlled by posttranslational modifications. While the role of phosphorylation in regulating GPCR signaling is well studied and established, the mechanisms by which other posttranslational modifications, such as ubiquitination, regulate GPCR signaling are not clearly defined. We hypothesize that GPCR ubiquitination and deubiquitination is critical for proper signaling and cellular responses. In the present study, we show that the deubiquitinase ubiquitin-specific protease-34 (USP34) regulates thrombin-stimulated protease-activated receptor-1 (PAR1)-induced p38 autophosphorylation and activation. The PAR1-stimulated p38 signaling pathway is driven by ubiquitination. Interestingly, small interfering RNA-induced knockdown of USP34 expression markedly increased PAR1 cell surface abundance and protein expression without modulating PAR1 ubiquitination or the ubiquitination status of p38 signaling pathway components. In addition, increased PAR1 expression observed in USP34-depleted cells was not caused by altered PAR1 constitutive internalization, agonist-induced internalization, or receptor degradation. Rather, we report that loss of USP34 expression increased mRNA transcript expression of the PAR1-encoding gene, F2R. This study unexpectedly identified a critical role for USP34 in regulation of F2R mRNA transcript expression, which modulates PAR1 cell surface levels and thrombin-stimulated p38 mitogen-activated protein kinase signaling.

Endothelial cells throughout the body sense blood flow, eliciting transcriptional and phenotypic responses. The brain endothelium, known as the blood-brain barrier (BBB), possesses unique barrier and transport properties, which are in part regulated by blood flow. We utilized RNA sequencing to analyze the transcriptome of primary cultured rat brain microvascular endothelial cells (BMECs), as well as three human induced pluripotent stem cell-derived models. We compared the transcriptional responses of these cells to either low (0.5 dyne/cm2) or high (12 dyne/cm2) shear stresses, and subsequent analyses identified genes and pathways that were influenced by shear including key BBB-associated genes (SLC2A1, LSR, PLVAP) and canonical endothelial shear-stress-response transcription factors (KLF2, KLF4). In addition, our analysis suggests that shear alone is insufficient to rescue the de-differentiation caused by in vitro primary BMEC culture. Overall, these datasets and analyses provide new insights into the influence of shear on BBB models that will aid in model selection and guide further model development.

We hypothesized that a strategy employing tissue-specific endothelial cells (EC) might facilitate the identification of tissue- or organ-specific vascular functions of ubiquitous metabolites. An unbiased approach was employed to identify water-soluble small molecules with mitogenic activity on choroidal EC. We identified adenosine diphosphate (ADP) as a candidate, following biochemical purification from mouse EL4 lymphoma extracts. ADP stimulated the growth of bovine choroidal EC (BCEC) and other bovine or human eye-derived EC. ADP induced rapid phosphorylation of extracellular signal-regulated kinase in a dose- and time-dependent manner. ADP-induced BCEC proliferation could be blocked by pretreatment with specific antagonists of the purinergic receptor P2Y1 but not with a vascular endothelial growth factor (VEGF) inhibitor, indicating that the EC mitogenic effects of ADP are not mediated by stimulation of the VEGF pathway. Intravitreal administration of ADP expanded the neovascular area in a mouse model of choroidal neovascularization. Single-cell transcriptomics from human choroidal datasets show the expression of P2RY1, but not other ADP receptors, in EC with a pattern similar to VEGFR2. Although ADP has been reported to be a growth inhibitor for vascular EC, here we describe its growth-stimulating effects for BCEC and other eye-derived EC.

Until now, Hippo pathway-mediated nucleocytoplasmic translocation has been considered the primary mechanism by which yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) transcriptional coactivators regulate cell proliferation and differentiation via transcriptional enhanced associate domain (TEAD)-mediated target gene expression. In this study, however, we found that TAZ, but not YAP, is associated with the Golgi apparatus in macrophages activated via Toll-like receptor ligands during the resolution phase of inflammation. Golgi-associated TAZ enhanced vesicle trafficking and secretion of proinflammatory cytokines in M1 macrophage independent of the Hippo pathway. Depletion of TAZ in tumor-associated macrophages promoted tumor growth by suppressing the recruitment of tumor-infiltrating lymphocytes. Moreover, in a diet-induced metabolic dysfunction-associated steatohepatitis model, macrophage-specific deletion of TAZ ameliorated liver inflammation and hepatic fibrosis. Thus, targeted therapies being developed against YAP/TAZ-TEAD are ineffective in macrophages. Together, our results introduce Golgi-associated TAZ as a potential molecular target for therapeutic intervention to treat tumor progression and chronic inflammatory diseases.

Eicosanoids are key players in inflammatory diseases and cancer. Targeting their production by inhibiting Group IVA cytosolic phospholipase A2 (cPLA2α) offers a promising approach for cancer therapy. In this study, we synthesize a second generation of thiazolyl ketone inhibitors of cPLA2α starting with compound GK470 (AVX235) and test their in vitro and cellular activities. We identify a more potent and selective lead molecule, GK420 (AVX420), which we test in parallel with AVX235 and a structurally unrelated compound, AVX002 for inhibition of cell viability across a panel of cancer cell lines. From this, we show that activity of polycomb group repressive complex 2 is a key molecular determinant of sensitivity to cPLA2α inhibition, while resistance depends on antioxidant response pathways. Consistent with these results, we show that elevated intracellular reactive oxygen species and activating transcription factor 4 target gene expression precede cell death in AVX420-sensitive T-cell acute lymphoblastic leukemia cells. Our findings imply cPLA2α may support cancer by mitigating oxidative stress and inhibiting tumor suppressor expression and suggest that AVX420 has potential for treating acute leukemias and other cancers that are susceptible to oxidative cell death.

Tumor initiation represents the first step in tumorigenesis during which normal progenitor cells undergo cell fate transition to cancer. Capturing this process as it occurs in vivo, however, remains elusive. Here we employ spatiotemporally controlled oncogene activation and tumor suppressor inhibition together with multiomics to unveil the processes underlying oral epithelial progenitor cell reprogramming into tumor initiating cells at single cell resolution. Tumor initiating cells displayed a distinct stem-like state, defined by aberrant proliferative, hypoxic, squamous differentiation, and partial epithelial to mesenchymal invasive gene programs. YAP-mediated tumor initiating cell programs included activation of oncogenic transcriptional networks and mTOR signaling, and recruitment of myeloid cells to the invasive front contributing to tumor infiltration. Tumor initiating cell transcriptional programs are conserved in human head and neck cancer and associated with poor patient survival. These findings illuminate processes underlying cancer initiation at single cell resolution, and identify candidate targets for early cancer detection and prevention.

Insulin is an important regulator of whole-body glucose homeostasis. In insulin sensitive tissues such as muscle and adipose, insulin induces the translocation of glucose transporter 4 (GLUT4) to the cell membrane, thereby increasing glucose uptake. However, insulin also signals in tissues that are not generally associated with glucose homeostasis. In the human reproductive endocrine axis, hyperinsulinemia suppresses the secretion of gonadotropins from gonadotrope cells of the anterior pituitary, thereby linking insulin dysregulation to suboptimal reproductive health. In the mouse, gonadotropes express the insulin receptor which has the canonical signaling response of IRS, AKT, and mTOR activation. However, the functional outcomes of insulin action on gonadotropes are unclear. Here, we demonstrate through use of an optimized cell fractionation protocol that insulin stimulation of the LβT2 gonadotropic cell line results in the unexpected translocation of GLUT1 to the plasma membrane. Using our high purity fractionation protocol, we further demonstrate that though Akt signaling in response to insulin is intact, insulin-induced translocation of GLUT1 occurs independently of Akt activation in LβT2 cells.