Adamts1 Mediates Ethanol‐Induced Alterations in Collagen and Elastin via a FoxO1‐Sestrin3‐AMPK Signaling Cascade in Myocytes

Abstract

A variety of stressors including alcohol (EtOH) are known to induce collagen production and fibrotic diseases. Matrix metalloproteinases (MMP) play an important role in regulating fibrosis, but little is known regarding the relationship between EtOH and MMPs. In addition, the signaling cascades involved in this process have not been elucidated. We have identified the MMP Adamts1 as a target of EtOH regulation. To characterize the function of Adamts1, we examined EtOH-induced alterations in collagen I and elastin protein levels in C2C12 myocytes. Incubation of myocytes with 100 mM EtOH decreased elastin and increased collagen content, respectively, and these changes were associated with increased O-GLcNAc modification of Adamts1. Conversely, silencing of Adamts1 by siRNA blocked the adverse effects of EtOH on collagen and elastin levels. Similar results were obtained after treatment with a pharmacological inhibitor of MMP. Changes in collagen were due, at least in part, to a decreased interaction of Adamts1 with its endogenous inhibitor TIMP3. The AMPK inhibitor compound C blocked the EtOH-induced stimulation of collagen and O-GLcNAc Adamts1 protein. Changes in AMPK appear linked to FoxO1, since inhibition of FoxO1 blocked the effects of EtOH on AMPK phosphorylation and O-GLcNAc levels. These FoxO-dependent modifications were associated with an upregulation of the FoxO1 transcription target sestrin 3, as well as increased binding of sestrin 3 with AMPK. Collectively, these data indicate that EtOH regulates the collagen I and elastin content in an Adamts1-dependent manner in myocytes. Furthermore, Adamts1 appears to be controlled by the FoxO1-sestrin 3-AMPK signaling cascade.

FoxO1‐AMPK‐ULK1 Regulates Ethanol‐Induced Autophagy in Muscle by Enhanced ATG14 Association with the BECN1‐PIK3C3 Complex

“…Previously, we reported that AMPK and FoxO1 mediate the effects of EtOH on various signal transduction pathways (Hong-Brown et al, 2015). As such, EtOH increases both AMPK and FoxO1 activity towards a number of downstream targets that regulate protein synthesis and extracellular matrix components.…”

“…We have previously reported that the FoxO1-sestrin3-AMPK signaling cascade mediates the ability of EtOH to inhibit mTOR function and protein synthesis in C2C12 myoblasts (Hong-Brown et al, 2015). Likewise, several studies have demonstrated a connection between AMPK and FoxO3a in the induction of autophagy, either under stress situations or following EtOH exposure (Nepal and Park, 2013, Ni et al, 2013, Sanchez et al, 2012).…”

“…All studies were conducted using cells at the early passage (P3–P7) of the myoblast stage. Upon confluence, cells were treated with 100 mM EtOH as previously described and harvested after 1 h unless otherwise indicated (Hong-Brown et al, 2015). A concentration of 100 mM EtOH was used because it produces an optimal effect on protein synthesis without being cytotoxic to myoblasts.…”

“…Furthermore, while AMPK regulates multiple PIK3C3 complexes following glucose deprivation (Kim et al, 2013), the role of AMPK in regulating PIK3C3 complexes as a mechanism underlying EtOH-induced autophagy in muscle is unknown. Previously, we reported EtOH enhances AMPK phosphorylation and activity, while concurrently inhibiting mTORC1 function and protein synthesis (Hong-Brown et al, 2010; Hong-Brown et al, 2015). Although recent studies showed that forkhead box 3a (FoxO3a) is a key factor in regulating EtOH and stress-induced autophagy in cultured hepatocytes and other tissues (Ni et al, 2013, Sanchez et al, 2012, Nepal and Park, 2013), it is unclear whether this signaling cascade regulates the process in muscle.…”

Alcohol Differentially Alters Extracellular Matrix and Adhesion Molecule Expression in Skeletal Muscle and Heart

“…Chronic alcohol consumption has been reported to increase TGFβ1 mRNA and protein in skeletal muscle (Clary et al, 2011). Moreover, in vitro studies indicate the alcohol-induced increase in collagen I protein in fibroblast conditioned medium (Law and Carver, 2013) and collagen α1(I) protein in myoblasts (Hong-Brown et al, 2015) was associated with increased TGF-β. Moreover, pretreatment with a TGFβ receptor inhibitor (SB 431542) or a soluble recombinant TGF-βII receptor prevented collagen accumulation suggesting the autocrine/paracrine actions of this cytokine were causally related to the profibrotic effect of alcohol (Law and Carver, 2013).…”