Biology subjects

Anthony, S. R.

Publications and source records attributed to Anthony, S. R..

5 recordsLinked to original sources

bioRxiv2026

WISP1 drives a mechanically active immune modulatory and proliferative cardiac myofibroblast state

Pathological cardiac remodeling is driven by the proliferation and differentiation of resident fibroblasts into active myofibroblasts and results in excessive extracellular matrix (ECM) deposition and tissue stiffening. Expression of the matricellular protein WISP1 has previously been shown to be increased with cardiac fibrosis and promote myofibroblast activity, but the mechanisms by which this occurs remain unknown. Primary cardiac fibroblasts were isolated from adult mouse hearts and treated with recombinant WISP1 or TGF{beta}1 both alone and in combination to determine the functional role of the matricellular protein WISP1 in driving cardiac myofibroblast activity. WISP1 significantly increased alpha-smooth muscle actin and collagen type I expression, total collagen secretion, collagen gel contractility, and wound healing equally in fibroblasts from both male and female mice. However, WISP1 alone failed to induce expression of periostin, a hallmark myofibroblast marker, suggesting the resulting WISP1-dependent cell phenotype is unique and/or acting through non-canonical pathways. Indeed, inhibition of P38 MAPK completely ablated the WISP1-dependent increase in SMA and collagen expression, while having little to no impact on TGF{beta}1-dependent expression of myofibroblast marker genes. We next employed a multi-omics approach to define the functional impact of WISP1 on fibroblast cell-state within the transcriptome, cytosolic, and secreted ECM proteome. RNA-seq results show that WISP1 broadly promotes the expression of proliferative and immune modulatory genes at the transcriptomic level, while having very little impact on traditional myofibroblast and ECM modifying gene expression programs. At the proteome level, WISP1 was again a much weaker mediator of traditional myofibroblast and ECM proteins. However, in agreement with RNA-seq data, we observed a strong WISP1-dependent enrichment for proliferation-associated proteins in the cytosolic proteome and inflammation-associated proteins in the ECM proteome. Interestingly, WISP1 also showed a context-dependent response with TGF{beta}1, suggesting a more complex and yet to be elucidated signaling interaction between these independent mediators of myofibroblast activity. In conclusion, our data suggests that WISP1 promotes a unique proliferative and immune-modulatory myofibroblast phenotype. HighlightsO_LIWISP1 is sufficient to drive myofibroblast SMA and collagen expression and ECM deposition C_LIO_LIWISP1 promotes canonical myofibroblast contractility and wound healing activity C_LIO_LIWISP1 mediates myofibroblast activity via a non-canonical, P38 MAPK-dependent signaling pathway C_LIO_LIMulti-omics analysis of WISP1-dependent RNA and protein expression show that WISP promotes a proliferative and immune modulatory myofibroblast phenotype C_LI

cell biology↗

bioRxiv2024

HuR-dependent expression of RyR2 contributes to calcium-mediated thermogenesis in brown adipocytes

Several uncoupling protein 1 (UCP1)-independent thermogenic pathways have been described in thermogenic adipose tissue, including calcium-mediated thermogenesis in beige adipocytes via sarco/endoplasmic reticulum ATPase (SERCA). We have previously shown that adipocyte-specific deletion of the RNA binding protein human antigen R (HuR) results in thermogenic dysfunction independent of UCP1 expression. RNA sequencing revealed the downregulation of several genes involved in calcium ion transport upon HuR deletion. The goal of this work was to define the HuR-dependent mechanisms of calcium driven thermogenesis in brown adipocytes. We generated (BAT)-specific HuR-deletion (BAT-HuR-/-) mice and show that their body weight, glucose tolerance, brown and white adipose tissue weights, and total lipid droplet size were not significantly different compared to wild-type. Similar to our initial findings in Adipo-HuR-/- mice, mice with BAT-specific HuR deletion are cold intolerant following acute thermal challenge at 4{degrees}C, demonstrating specificity of acute HuR-dependent thermogenesis to BAT. We also found decreased expression of ryanodine receptor 2 (RyR2), but no changes in RyR2, SERCA1, SERCA2, or UCP1 expression, in BAT from BAT-HuR-/- mice. Next, we used Fluo-4 calcium indicator dye to show that genetic deletion or pharmacological inhibition of HuR blunts the increase in cytosolic calcium concentration in SVF-derived primary brown adipocytes. Moreover, we saw a similar blunting in {beta}-adrenergic-mediated heat generation, as assessed by ERtherm AC fluorescence, in SVF-derived brown adipocytes following HuR inhibition or deletion. Mechanistically, we show that HuR directly binds and reduces the decay rate of RyR2 mRNA in brown adipocytes, and stabilization of RyR2 via S107 rescues {beta}-adrenergic-mediated cytosolic calcium increase and heat generation in HuR deficient brown adipocytes. In conclusion, our results suggest that HuR-dependent control of RyR2 expression plays a significant role in the thermogenic function of brown adipose tissue through modulation of SR calcium cycling.

molecular biology↗

bioRxiv2023

HuR inhibition reduces post-ischemic cardiac remodeling by dampening acute inflammatory gene expression and the innate immune response

Myocardial ischemia/reperfusion (I/R) injury and the resulting cardiac remodeling is a common cause of heart failure. The RNA binding protein Human Antigen R (HuR) has been previously shown to reduce cardiac remodeling following both I/R and cardiac pressure overload, but the full extent of the HuR-dependent mechanisms within cells of the myocardium have yet to be elucidated. In this study, we applied a novel small molecule inhibitor of HuR to define the functional role of HuR in the acute response to I/R injury and gain a better understanding of the HuR-dependent mechanisms during post-ischemic myocardial remodeling. Our results show an early (two hours post-I/R) increase in HuR activity that is necessary for early inflammatory gene expression by cardiomyocytes in response to I/R. Surprisingly, despite the reductions in early inflammatory gene expression at two hours post-I/R, HuR inhibition has no effect on initial infarct size at 24-hours post-I/R. However, in agreement with previously published work, we do see a reduction in pathological remodeling and preserved cardiac function at two weeks post-I/R upon HuR inhibition. RNA-sequencing analysis of neonatal rat ventricular myocytes (NRVMs) at two hours post-LPS treatment to model damage associated molecular pattern (DAMP)-mediated activation of toll like receptors (TLRs) demonstrates a broad HuR-dependent regulation of pro-inflammatory chemokine and cytokine gene expression in cardiomyocytes. We show that conditioned media from NRVMs pre-treated with HuR inhibitor loses the ability to induce inflammatory gene expression in bone marrow derived macrophages (BMDMs) compared to NRVMs treated with LPS alone. Functionally, HuR inhibition in NRVMs also reduces their ability to induce endocrine migration of peripheral blood monocytes in vitro and reduces post-ischemic macrophage infiltration to the heart in vivo. In summary, these results suggest a HuR-dependent expression of pro-inflammatory gene expression by cardiomyocytes that leads to subsequent monocyte recruitment and macrophage activation in the post-ischemic myocardium.

molecular biology↗

bioRxiv2022

HuR-dependent expression of Wisp1 is necessary for TGF????-induced cardiac myofibroblast activity

Cardiac fibrosis is regulated by the activation and phenotypic switching of quiescent cardiac fibroblasts to active myofibroblasts, which have extracellular matrix (ECM) remodeling and contractile functions which play a central role in cardiac remodeling in response to injury. Here, we show that expression and activity of the RNA binding protein HuR is increased in cardiac fibroblasts upon transformation to an active myofibroblast. Pharmacological inhibition of HuR significantly blunts the TGF{beta}-dependent increase in ECM remodeling genes, total collagen secretion, in vitro scratch closure, and collagen gel contraction in isolated primary cardiac fibroblasts, suggesting a suppression of TGF{beta}-induced myofibroblast activation upon HuR inhibition. To delineate HuR-dependent mechanisms, we used photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) to identify eleven mRNA transcripts that showed enriched HuR binding following TGF{beta} treatment as well as significant co-expression correlation with HuR, SMA, and periostin using single-cell RNA-sequencing from ischemic-zone isolated fibroblasts. Of these, Wnt1-inducible signaling pathway protein-1 (Wisp1; Ccn4), was the most significantly associated with HuR expression in fibroblasts. Accordingly, we found Wisp1 expression to be increased in cardiac fibroblasts isolated from the ischemic-zone of mouse hearts following ischemia/reperfusion, and confirmed Wisp1 expression to be HuR-dependent in isolated fibroblasts. Finally, addition of exogenous recombinant Wisp1 is able to partially rescue myofibroblast contractile function following HuR inhibition, demonstrating that HuR-dependent Wisp1 expression plays a functional role in HuR-dependent MF activity downstream of TGF{beta}. In conclusion, HuR activity is necessary for the functional activation of primary cardiac fibroblasts in response to TGF{beta}, in part through post-transcriptional regulation of Wisp1. HIGHLIGHTSO_LIThe RNA binding protein HuR is highly expressed in cardiac fibroblasts and its expression strongly correlates with markers of active myofibroblasts C_LIO_LIHuR inhibition reduces migration, contraction, and ECM production activity of cardiac fibroblasts C_LIO_LIExpression of the secreted matricellular protein, Wisp1, is increased in a HuR-dependent manner following TGF{beta} treatment C_LIO_LIRecombinant Wisp1 rescues myofibroblast contractile function following HuR inhibition C_LIO_LIHuR-dependent expression of Wisp1 is necessary for myofibroblast activation C_LI

molecular biology↗

bioRxiv2021

Adipocyte deletion of the RNA binding protein HuR induces cardiac hypertrophy and fibrosis

Adipose tissue continues to gain appreciation for its broad role as an endocrine organ, and disruptions in adipose tissue homeostasis plays a central role in cardiovascular physiology. We have previously shown that expression of the RNA binding protein HuR in adipose tissue mediates energy expenditure, but the potential cardiovascular impacts of this finding have not been explored. We show here that adipose tissue-specific deletion of HuR (Adipo-HuR-/-) is sufficient to induce the spontaneous development of cardiac hypertrophy and fibrosis. Hearts from Adipo-HuR-/- mice have increased left ventricular (LV) ejection fraction, rate of pressure generation, and LV posterior wall thickness that is accompanied by an increase in LV/body weight ratio and hypertrophic gene expression. Furthermore, Adipo-HuR-/- hearts display increased fibrosis by picrosirius red staining and periostin expression. To identify underlying mechanisms, we applied both RNA-seq and weighted gene co-expression network analysis (WGCNA) to define HuR-dependent changes in gene expression as well as significant relationships between adipose tissue gene expression and LV mass. RNA-seq results demonstrate a significant increase in pro-inflammatory gene expression in the subcutaneous white adipose tissue (scWAT) from Adipo-HuR-/- mice that is accompanied by an increase in serum levels of both TNF- and IL-6. WGCNA identified a significant enrichment in inflammation, apoptosis/cell death, and vesicle-mediated transport genes among those whose expression most significantly associated with CVD in Adipo-HuR-/-. In conclusion, we demonstrate that the loss of HuR expression in adipose tissue promotes the development of cardiac hypertrophy and fibrosis, potentially through modulation of inflammation and vesicle-mediated transport in scWAT. NEW AND NOTEWORTHYThis work demonstrates the spontaneous development of cardiac hypertrophy and fibrosis upon adipose tissue-specific deletion of the RNA binding protein HuR that appears to be mechanistically driven by HuR-dependent changes in inflammatory and extracellular vesicle transport mediating genes in the subcutaneous white adipose tissue. These results suggest that loss of HuR expression in adipose tissue in obesity, as demonstrated in mouse and humans by our group and others, may contribute to obesity-mediated CVD.

physiology↗