Search

Heart failure with preserved ejection fraction (HFpEF) is a widespread syndrome with limited therapeutic options and poorly understood immune pathophysiology. Using a 2-hit preclinical model of cardiometabolic HFpEF that induces obesity and hypertension, we found that cardiac T cell infiltration and lymphoid expansion occurred concomitantly with cardiac pathology and that diastolic dysfunction, cardiomyocyte hypertrophy, and cardiac phospholamban phosphorylation were T cell dependent. Heart-infiltrating T cells were not restricted to cardiac antigens and were uniquely characterized by impaired activation of the inositol-requiring enzyme 1[alpha]/X-box-binding protein 1 (IRE1[alpha]/XBP1) arm of the unfolded protein response. Notably, selective ablation of XBP1 in T cells enhanced their persistence in the heart and lymphoid organs of mice with preclinical HFpEF. Furthermore, T cell IRE1[alpha]/XBP1 activation was restored after withdrawal of the 2 comorbidities inducing HFpEF, resulting in partial improvement of cardiac pathology. Our results demonstrated that diastolic dysfunction and cardiomyocyte hypertrophy in preclinical HFpEF were T cell dependent and that reversible dysregulation of the T cell IRE1[alpha]/ XBP1 axis was a T cell signature of HFpEF., Introduction Widely regarded as the greatest unmet need in cardiovascular medicine, heart failure with preserved ejection fraction (HFpEF) is a complex and heterogeneous syndrome accounting for roughly 50% of HF [...]

A cascade of three enzymes, E1−E2−E3, is responsible for transferring ubiquitin to target proteins, which controls many different aspects of cellular signaling. The role of the E2 has been largely overlooked, despite influencing substrate identity, chain multiplicity, and topology. Here we report a method—targeted charging of ubiquitin to E2 (tCUbE)—that can track a tagged ubiquitin through its entire enzymatic cascade in living mammalian cells. We use this approach to reveal new targets whose ubiquitination depends on UbcH5a E2 activity. We demonstrate that tCUbE can be broadly applied to multiple E2s and in different human cell lines. tCUbE is uniquely suited to examine E2−E3‐substrate cascades of interest and/or piece together previously unidentified cascades, thereby illuminating entire branches of the UPS and providing critical insight that will be useful for identifying new therapeutic targets in the UPS. [ABSTRACT FROM AUTHOR]

Simple Summary: Skin cancer, such as melanoma, is often treatable but becomes deadly when it expands to other places in the body, a process called metastasis. While many current drugs target melanoma tumor growth, no drugs yet exist to specifically prevent metastasis. Improving treatment outcomes, therefore, will require an understanding of the molecular mechanisms leading to metastasis. The AKT family of proteins are important regulators of cellular growth and signaling, which play differing roles in cancer. This work sought to study each of the AKT isoforms and their contributions to melanoma cell migration and metastasis. We found that AKT2 specifically regulates melanoma cell metastasis through effects on metabolism and melanoma cell properties, while AKT1 is involved in cellular proliferation and growth. This study suggests that specifically targeting AKT2 and AKT1 represents novel therapeutic strategies for different-stage melanoma patients. Despite recent advances in treatment, melanoma remains the deadliest form of skin cancer due to its highly metastatic nature. Melanomas harboring oncogenic BRAFV600E mutations combined with PTEN loss exhibit unrestrained PI3K/AKT signaling and increased invasiveness. However, the contribution of different AKT isoforms to melanoma initiation, progression, and metastasis has not been comprehensively explored, and questions remain about whether individual isoforms play distinct or redundant roles in each step. We investigate the contribution of individual AKT isoforms to melanoma initiation using a novel mouse model of AKT isoform-specific loss in a murine melanoma model, and we investigate tumor progression, maintenance, and metastasis among a panel of human metastatic melanoma cell lines using AKT isoform-specific knockdown studies. We elucidate that AKT2 is dispensable for primary tumor formation but promotes migration and invasion in vitro and metastatic seeding in vivo, whereas AKT1 is uniquely important for melanoma initiation and cell proliferation. We propose a mechanism whereby the inhibition of AKT2 impairs glycolysis and reduces an EMT-related gene expression signature in PTEN-null BRAF-mutant human melanoma cells to limit metastatic spread. Our data suggest that the elucidation of AKT2-specific functions in metastasis might inform therapeutic strategies to improve treatment options for melanoma patients. [ABSTRACT FROM AUTHOR]

R3436 --> 10.4 --> Heart failure (HF) is a leading cause of death worldwide, associated with cardiac and systemic inflammation. However, no anti‐inflammatory therapies have shown success. Damage associated molecular patterns (DAMPs) released in the heart activate myeloid cells through the adaptor "Myeloid differentiation primary response 88" (MyD88). This signaling cascade promotes cardiac antigen presentation to activate helper T‐cells. Once activated, T‐cells then infiltrate the heart and bind to VCAM‐1 on cardiac fibroblasts, (CFB), transforming them to myofibroblasts which coordinate cardiac fibrosis. MyD88 is also expressed in T‐cells, yet whether it is pro or anti‐inflammatory is less explored, and evidence suggests is context dependent. To investigate the role of T‐cell MyD88 in HF, we reconstituted Tcra‐/‐ mice, normally protected from HF induced by transaortic constriction (TAC), with WT or Myd88‐/‐ Type 1 helper T‐cells (Th1) in the onset of TAC. Surprisingly, we found that mice receiving Myd88‐/‐ Th1 cells showed increased cardiac T‐cells and fibrosis compared to mice receiving WT cells. We hypothesized that T‐cell MyD88 regulates cardiac T‐cell recruitment and survival, and as a result subsequent cardiac fibrosis. We found that Myd88‐/‐ Th1 cells exhibited increased survival in vitro determined by real time imaging of propidium iodide incorporation, as well as in vivo by performing competitive survival studies in which Tcra‐/‐ mice received equal WT CD45.1 Th1 cells and Myd88‐/‐ CD45.2 Th1 cells. Myd88‐/‐ Th1 cells also induced higher CFB α‐smooth muscle actin staining when cultured together, as well as bound more frequently to CFB and immobilized VCAM‐1. Together these data demonstrate that MyD88 limits T‐cell mediated cardiac fibrosis by regulating T‐cell survival, as well as by dampening T‐cell/fibroblast interactions. We identify novel role for T‐cell MyD88 in cardiac inflammation that may be modulated in HF. [ABSTRACT FROM AUTHOR]

Background: Cardiac inflammation in heart failure is characterized by the presence of damage-associated molecular patterns, myeloid cells, and T cells. Cardiac damage-associated molecular patterns provide continuous proinflammatory signals to myeloid cells through TLRs (toll-like receptors) that converge onto the adaptor protein MyD88 (myeloid differentiation response 88). These induce activation into efficient antigen-presenting cells that activate T cells through their TCR (T-cell receptor). T-cell activation results in cardiotropism, cardiac fibroblast transformation, and maladaptive cardiac remodeling. T cells rely on TCR signaling for effector function and survival, and while they express MyD88 and damage-associated molecular pattern receptors, their role in T-cell activation and cardiac inflammation is unknown., Methods: We performed transverse aortic constriction in mice lacking MyD88 in T cells and analyzed remodeling, systolic function, survival, and T-cell activation. We profiled wild type versus Myd88 -/- mouse T cells at the transcript and protein level and performed several functional assays., Results: Analysis of single-cell RNA-sequencing data sets revealed that MyD88 is expressed in mouse and human cardiac T cells. MyD88 deletion in T cells resulted in increased levels of cardiac T-cell infiltration and fibrosis in response to transverse aortic constriction. We discovered that TCR-activated Myd88 -/- T cells had increased proinflammatory signaling at the transcript and protein level compared with wild type, resulting in increased T-cell effector functions such as adhesion, migration across endothelial cells, and activation of cardiac fibroblast. Mechanistically, we found that MyD88 modulates T-cell activation and survival through TCR-dependent rather than TLR-dependent signaling., Conclusions: Our results outline a novel intrinsic role for MyD88 in limiting T-cell activation that is central to tune down cardiac inflammation during cardiac adaptation to stress., Competing Interests: Disclosures None.

Cellular senescence is a carefully regulated process of proliferative arrest accompanied by functional and morphologic changes. Senescence allows damaged cells to avoid neoplastic proliferation; however, the induction of the senescence-associated secretory phenotype (SASP) can promote tumor growth. The complexity of senescence may limit the efficacy of anti-neoplastic agents, such as CDK4/6 inhibitors (Cdk4/6i), that induce a senescence-like state in tumor cells. The AKT kinase family, which contains three isoforms that play both unique and redundant roles in cancer progression, is commonly hyperactive in many cancers including melanoma and has been implicated in the regulation of senescence. To interrogate the role of AKT isoforms in Cdk4/6i-induced cellular senescence, we generated isoform-specific AKT knockout human melanoma cell lines. We found that the CDK4/6i Palbociclib induced a form of senescence in these cells that was dependent on AKT1. We then evaluated the activity of the cGAS-STING pathway, recently implicated in cellular senescence, finding that cGAS-STING function was dependent on AKT1, and pharmacologic inhibition of cGAS had little effect on senescence. However, we found SASP factors to require NF-κB function, in part dependent on a stimulatory phosphorylation of IKKα by AKT1. In summary, we provide the first evidence of a novel, isoform-specific role for AKT1 in therapy-induced senescence in human melanoma cells acting through NF-κB but independent of cGAS.

The stimulator of IFN genes (STING) protein senses cyclic dinucleotides released in response to double-stranded DNA and functions as an adaptor molecule for type I IFN (IFNI) signaling by activating IFNI-stimulated genes (ISG). We found impaired T cell infiltration into the peritoneum in response to TNF-α in global and EC-specific STING-/- mice and discovered that T cell transendothelial migration (TEM) across mouse and human endothelial cells (EC) deficient in STING was strikingly reduced compared with control EC, whereas T cell adhesion was not impaired. STING-/- T cells showed no defect in TEM or adhesion to EC, or immobilized endothelial cell-expressed molecules ICAM1 and VCAM1, compared with WT T cells. Mechanistically, CXCL10, an ISG and a chemoattractant for T cells, was dramatically reduced in TNF-α-stimulated STING-/- EC, and genetic loss or pharmacologic antagonisms of IFNI receptor (IFNAR) pathway reduced T cell TEM. Our data demonstrate a central role for EC-STING during T cell TEM that is dependent on the ISG CXCL10 and on IFNI/IFNAR signaling.