Catherine E. Winbanks

miR-200a Prevents Renal Fibrogenesis Through Repression of TGF-β2 Expression

OBJECTIVE Progressive fibrosis in the diabetic kidney is driven and sustained by a diverse range of profibrotic factors. This study examines the critical role of microRNAs (miRNAs) in the regulation of the key fibrotic mediators, TGF-β1 and TGF-β2. RESEARCH DESIGN AND METHODS Rat proximal-tubular epithelial cells (NRK52E) were treated with TGF-β1 and TGF-β2 for 3 days, and expression of markers of epithelial-to-mesenchymal transition (EMT) and fibrogenesis were assessed by RT-PCR and Western blotting. The expression of miR-141 and miR-200a was also assessed, as was their role as translational repressors of TGF-β signaling. Finally, these pathways were explored in two different mouse models, representing early and advanced diabetic nephropathy. RESULTS Both TGF-β1 and TGF-β2 induced EMT and fibrogenesis in NRK52E cells. TGF-β1 and TGF-β2 also downregulated expression of miR-200a. The importance of these changes was demonstrated by the finding that ectopic expression miR-200a downregulated smad-3 activity and the expression of matrix proteins and prevented TGF-β–dependent EMT. miR-200a also downregulated the expression of TGF-β2, via direct interaction with the 3′ untranslated region of TGF-β2. The renal expression of miR-141 and miR-200a was also reduced in mouse models representing early and advanced kidney disease. CONCLUSIONS miR-200a and miR-141 significantly impact on the development and progression of TGF-β–dependent EMT and fibrosis in vitro and in vivo. These miRNAs appear to be intricately involved in fibrogenesis, both as downstream mediators of TGF-β signaling and as components of feedback regulation, and as such represent important new targets for the prevention of progressive kidney disease in the context of diabetes.

Therapeutic inhibition of the miR-34 family attenuates pathological cardiac remodeling and improves heart function

MicroRNAs are dysregulated in a setting of heart disease and have emerged as promising therapeutic targets. MicroRNA-34 family members (miR-34a, -34b, and -34c) are up-regulated in the heart in response to stress. In this study, we assessed whether inhibition of the miR-34 family using an s.c.-delivered seed-targeting 8-mer locked nucleic acid (LNA)-modified antimiR (LNA-antimiR-34) can provide therapeutic benefit in mice with preexisting pathological cardiac remodeling and dysfunction due to myocardial infarction (MI) or pressure overload via transverse aortic constriction (TAC). An additional cohort of mice subjected to MI was given LNA-antimiR-34a (15-mer) to inhibit miR-34a alone as a comparison for LNA-antimiR-34. LNA-antimiR-34 (8-mer) efficiently silenced all three miR-34 family members in both cardiac stress models and attenuated cardiac remodeling and atrial enlargement. In contrast, inhibition of miR-34a alone with LNA-antimiR-34a (15-mer) provided no benefit in the MI model. In mice subjected to pressure overload, LNA-antimiR-34 improved systolic function and attenuated lung congestion, associated with reduced cardiac fibrosis, increased angiogenesis, increased Akt activity, decreased atrial natriuretic peptide gene expression, and maintenance of sarcoplasmic reticulum Ca2+ ATPase gene expression. Improved outcome in LNA-antimiR-34–treated MI and TAC mice was accompanied by up-regulation of several direct miR-34 targets, including vascular endothelial growth factors, vinculin, protein O-fucosyltranferase 1, Notch1, and semaphorin 4B. Our results provide evidence that silencing of the entire miR-34 family can protect the heart against pathological cardiac remodeling and improve function. Furthermore, these data underscore the utility of seed-targeting 8-mer LNA-antimiRs in the development of new therapeutic approaches for pharmacologic inhibition of disease-implicated miRNA seed families.

TGF-β regulates miR-206 and miR-29 to control myogenic differentiation through regulation of HDAC4

MicroRNAs (miRs) are emerging as prominent players in the regulation of many biological processes, including myogenic commitment and skeletal muscle formation. Members of the TGF-β family can influence the proliferation and myogenic differentiation of cells, although it is presently not clear what role miRNAs play in the TGF-β-mediated control of myogenic differentiation. Here, we demonstrate in the myogenic C2C12 cell line, and in primary muscle cells, that miR-206 and miR-29-two miRs that act on transcriptional events implicated in muscle differentiation are down-regulated by TGF-β. We further demonstrate that TGF-β treatment of myogenic cells is associated with increased expression of histone deacetylase 4 (HDAC4), a key inhibitor of muscle differentiation that has been identified as a target for regulation by miR-206 and miR-29. We confirmed that increased expression of miR-206 and miR-29 resulted in the translational repression of HDAC4 in the presence or absence of TGF-β via interaction with the HDAC4 3′-untranslated region. Importantly, we found that miR-206 and miR-29 can attenuate the inhibitory actions of TGF-β on myogenic differentiation. Furthermore, we present evidence that the mechanism by which miR-206 and miR-29 can inhibit the TGF-β-mediated up-regulation of HDAC4 is via the inhibition of Smad3 expression, a transducer of TGF-β signaling. These findings identify a novel mechanism of interaction between TGF-β and miR-206 and -29 in the regulation of myogenic differentiation through HDAC4.

Follistatin-mediated skeletal muscle hypertrophy is regulated by Smad3 and mTOR independently of myostatin

Smad3/Akt/mTOR/S6K/S6RP signaling plays a critical role in follistatin-mediated muscle growth that operates independently of myostatin-driven mechanisms.

Elevated expression of activins promotes muscle wasting and cachexia

In models of cancer cachexia, inhibiting type IIB activin receptors (ActRIIBs) reverse muscle wasting and prolongs survival, even with continued tumor growth. ActRIIB mediates signaling of numerous TGF‐β proteins; of these, we demonstrate that activins are the most potent negative regulators of muscle mass. To determine whether activin signaling in the absence of tumor‐derived factors induces cachexia, we used recombinant serotype 6 adeno‐associated virus (rAAV6) vectors to increase circulating activin A levels in C57BL/6 mice. While mice injected with control vector gained ~10% of their starting body mass (3.8±0.4 g) over 10 wk, mice injected with increasing doses of rAAV6:activin A exhibited weight loss in a dose‐dependent manner, to a maximum of –12.4% (–4.2±1.1 g). These reductions in body mass in rAAV6:activin‐injected mice correlated inversely with elevated serum activin A levels (7‐ to 24‐fold). Mechanistically, we show that activin A reduces muscle mass and function by stimulating the ActRIIB pathway, leading to deleterious consequences, including increased transcription of atrophy‐related ubiquitin ligases, decreased Akt/mTOR‐mediated protein synthesis, and a profibrotic response. Critically, we demonstrate that the muscle wasting and fibrosis that ensues in response to excessive activin levels is fully reversible. These findings highlight the therapeutic potential of targeting activins in cachexia.—Chen, J. L., Walton, K. L., Winbanks, C. E., Murphy, K. T., Thomson, R. E., Makanji, Y., Qian, H., Lynch, G. S., Harrison, C. A., Gregorevic, P. Elevated expression of activins promotes muscle wasting and cachexia. FASEB J. 28, 28–1711 (1723). www.fasebj.org

The bone morphogenetic protein axis is a positive regulator of skeletal muscle mass

The BMP signaling pathway promotes muscle growth and inhibits muscle wasting via SMAD1/5-dependent signaling.

miR-21 promotes renal fibrosis in diabetic nephropathy by targeting PTEN and SMAD7.

The cytokine transforming growth factor (TGF)-β1 plays a central role in diabetic nephropathy (DN) with data implicating the miRNA (miR) miR-21 as a key modulator of its prosclerotic actions. In the present study, we demonstrate data indicating that miR-21 up-regulation positively correlates with the severity of fibrosis and rate of decline in renal function in human DN. Furthermore, concomitant analyses of various models of fibrotic renal disease and experimental DN, confirm tubular miR-21 up-regulation. The fibrotic changes associated with increased miR-21 levels are proposed to include the regulation of TGF-β1-mediated mothers against decapentaplegic homolog 3 (SMAD3)- and phosphoinositide 3-kinase (PI3K)-dependent signalling pathways via co-ordinated repression of mothers against decapentaplegic homolog 7 (SMAD7) and phosphatase and tensin homologue (PTEN) respectively. This represents a previously uncharacterized interaction axis between miR-21 and PTEN-SMAD7. Targeting of these proteins by miR-21 resulted in de-repression of the respective pathways as reflected by increases in SMAD3 and V-Akt murine thymoma viral oncogene homolog 1 (AKT) phosphorylation. Many of the changes typically induced by TGF-β1, including phosphorylation of signalling mediators, were further enhanced by miR-21. Collectively, these data present a unified model for a key role for miR-21 in the regulation of renal tubular extracellular matrix (ECM) synthesis and accumulation and provide important insights into the molecular pathways implicated in the progression of DN.

miR-206 represses hypertrophy of myogenic cells but not muscle fibers via inhibition of HDAC4.

microRNAs regulate the development of myogenic progenitors, and the formation of skeletal muscle fibers. However, the role miRNAs play in controlling the growth and adaptation of post-mitotic musculature is less clear. Here, we show that inhibition of the established pro-myogenic regulator miR-206 can promote hypertrophy and increased protein synthesis in post-mitotic cells of the myogenic lineage. We have previously demonstrated that histone deacetylase 4 (HDAC4) is a target of miR-206 in the regulation of myogenic differentiation. We confirmed that inhibition of miR-206 de-repressed HDAC4 accumulation in cultured myotubes. Importantly, inhibition of HDAC4 activity by valproic acid or sodium butyrate prevented hypertrophy of myogenic cells otherwise induced by inhibition of miR-206. To test the significance of miRNA-206 as a regulator of skeletal muscle mass in vivo, we designed recombinant adeno-associated viral vectors (rAAV6 vectors) expressing miR-206, or a miR-206 “sponge,” featuring repeats of a validated miR-206 target sequence. We observed that over-expression or inhibition of miR-206 in the muscles of mice decreased or increased endogenous HDAC4 levels respectively, but did not alter muscle mass or myofiber size. We subsequently manipulated miR-206 levels in muscles undergoing follistatin-induced hypertrophy or denervation-induced atrophy (models of muscle adaptation where endogenous miR-206 expression is altered). Vector-mediated manipulation of miR-206 activity in these models of cell growth and wasting did not alter gain or loss of muscle mass respectively. Our data demonstrate that although the miR-206/HDAC4 axis operates in skeletal muscle, the post-natal expression of miR-206 is not a key regulator of basal skeletal muscle mass or specific modes of muscle growth and wasting. These studies support a context-dependent role of miR-206 in regulating hypertrophy that may be dispensable for maintaining or modifying the adult skeletal muscle phenotype – an important consideration in relation to the development of therapeutics designed to manipulate microRNA activity in musculature.

Therapeutic silencing of miR-652 restores heart function and attenuates adverse remodeling in a setting of established pathological hypertrophy

Expression of microRNA‐652 (miR‐652) increases in the diseased heart, decreases in a setting of cardioprotection, and is inversely correlated with heart function. The aim of this study was to assess the therapeutic potential of inhibiting miR‐652 in a mouse model with established pathological hypertrophy and cardiac dysfunction due to pressure overload. Mice were subjected to a sham operation or transverse aortic constriction (TAC) for 4 wk to induce hypertrophy and cardiac dysfunction, followed by administration of a locked nucleic acid (LNA)‐antimiR‐652 (miR‐652 inhibitor) or LNA control. Cardiac function was assessed before and 8 wk post‐treatment. Expression of miR‐652 increased in hearts subjected to TAC compared to sham surgery (2.9‐fold), and this was suppressed by ~95% in LNA‐antimiR‐652‐treated TAC mice. Inhibition of miR‐652 improved cardiac function in TAC mice (fractional shortening:29±1% at 4 wk post‐TAC compared to 35±1% post‐treatment) and attenuated cardiac hypertrophy. Improvement in heart function was associated with reduced cardiac fibrosis, less apoptosis and B‐type natriuretic peptide gene expression, and preserved angiogenesis. Mechanistically, we identified Jagged1 (a Notch1 ligand) as a novel direct target of miR‐652. In summary, these studies provide the first evidence that silencing of miR‐652 protects the heart against pathological remodeling and improves heart function.—Bernardo, B. C., Nguyen, S. S., Winbanks, C. E., Gao, X.‐M., Boey, E. J. H., Tham, Y. K., Kiriazis, H., Ooi, J. Y. Y., Porrello, E. R., Igoor, S., Thomas, C. J., Gregorevic, P., Lin, R. C. Y., Du, X.‐J., McMullen, J. R. Therapeutic silencing of miR‐652 restores heart function and attenuates adverse remodeling in a setting of established pathological hypertrophy. FASEB J. 28, 5097–5110 (2014). www.fasebj.org

Silencing of miR-34a Attenuates Cardiac Dysfunction in a Setting of Moderate, but Not Severe, Hypertrophic Cardiomyopathy

Therapeutic inhibition of the miR-34 family (miR-34a,-b,-c), or miR-34a alone, have emerged as promising strategies for the treatment of cardiac pathology. However, before advancing these approaches further for potential entry into the clinic, a more comprehensive assessment of the therapeutic potential of inhibiting miR-34a is required for two key reasons. First, miR-34a has ∼40% fewer predicted targets than the miR-34 family. Hence, in cardiac stress settings in which inhibition of miR-34a provides adequate protection, this approach is likely to result in less potential off-target effects. Secondly, silencing of miR-34a alone may be insufficient in settings of established cardiac pathology. We recently demonstrated that inhibition of the miR-34 family, but not miR-34a alone, provided benefit in a chronic model of myocardial infarction. Inhibition of miR-34 also attenuated cardiac remodeling and improved heart function following pressure overload, however, silencing of miR-34a alone was not examined. The aim of this study was to assess whether inhibition of miR-34a could attenuate cardiac remodeling in a mouse model with pre-existing pathological hypertrophy. Mice were subjected to pressure overload via constriction of the transverse aorta for four weeks and echocardiography was performed to confirm left ventricular hypertrophy and systolic dysfunction. After four weeks of pressure overload (before treatment), two distinct groups of animals became apparent: (1) mice with moderate pathology (fractional shortening decreased ∼20%) and (2) mice with severe pathology (fractional shortening decreased ∼37%). Mice were administered locked nucleic acid (LNA)-antimiR-34a or LNA-control with an eight week follow-up. Inhibition of miR-34a in mice with moderate cardiac pathology attenuated atrial enlargement and maintained cardiac function, but had no significant effect on fetal gene expression or cardiac fibrosis. Inhibition of miR-34a in mice with severe pathology provided no therapeutic benefit. Thus, therapies that inhibit miR-34a alone may have limited potential in settings of established cardiac pathology.