O. Alvarez-Garcia

FoxO Transcription Factors Support Oxidative Stress Resistance in Human Chondrocytes

A major signaling pathway that regulates cellular aging is the insulin/insulin‐like growth factor 1 (IGF‐1)/phosphatidylinositol 3‐kinase (PI3K)/Akt/FoxO transcription factor axis. We previously observed that FoxO transcription factors are dysregulated in aged and OA cartilage. The objective of this study was to investigate the impact of down‐regulated FoxO transcription factors on chondrocytes.

Palmitate Has Proapoptotic and Proinflammatory Effects on Articular Cartilage and Synergizes With Interleukin-1

Obesity is a major risk factor for the development of osteoarthritis (OA) that is associated with a state of low‐grade inflammation and increased circulating levels of adipokines and free fatty acids (FFAs). The aim of this study was to analyze the effects of saturated (palmitate) and monounsaturated (oleate) FFAs on articular chondrocytes, synoviocytes, and cartilage.

Antisense RNA Controls LRP1 Sense Transcript Expression through Interaction with a Chromatin-Associated Protein, HMGB2

Long non-coding RNAs (lncRNAs), including natural antisense transcripts (NATs), are expressed more extensively than previously anticipated and have widespread roles in regulating gene expression. Nevertheless, the molecular mechanisms of action of the majority of NATs remain largely unknown. Here, we identify a NAT of low-density lipoprotein receptor-related protein 1 (Lrp1), referred to as Lrp1-AS, that negatively regulates Lrp1 expression. We show that Lrp1-AS directly binds to high-mobility group box 2 (Hmgb2) and inhibits the activity of Hmgb2 to enhance Srebp1a-dependent transcription of Lrp1. Short oligonucleotides targeting Lrp1-AS inhibit the interaction of antisense transcript and Hmgb2 protein and increase Lrp1 expression by enhancing Hmgb2 activity. Quantitative RT-PCR analysis of brain tissue samples from Alzheimers disease patients and aged-matched controls revealed upregulation of LRP1-AS and downregulation of LRP1. Our data suggest a regulatory mechanism whereby a NAT interacts with a ubiquitous chromatin-associated protein to modulate its activity in a locus-specific fashion.

Suppression of REDD1 in osteoarthritis cartilage, a novel mechanism for dysregulated mTOR signaling and defective autophagy

OBJECTIVE Aging is a main risk factor for the development of osteoarthritis (OA) and the molecular mechanisms underlying the aging-related changes in articular cartilage include increased mammalian target of rapamycin (mTOR) signaling and defective autophagy. REDD1 is an endogenous inhibitor of mTOR that regulates cellular stress responses. In this study we measured REDD1 expression in normal, aged and OA cartilage and assessed REDD1 function in human and mouse articular chondrocytes. METHODS REDD1 expression was analyzed in human and mouse articular cartilage by qPCR, western blotting, and immunohistochemistry. For functional studies, REDD1 and TXNIP knockdown or overexpression was performed in chondrocytes in the presence or absence of rapamycin and chloroquine, and mTOR signaling and autophagy were measured by western blotting. REDD1/TXNIP protein interaction was assessed by co-immunoprecipitation experiments. RESULTS Human and mouse cartilage from normal knee joints expressed high levels of REDD1. REDD1 expression was significantly reduced in aged and OA cartilage. In cultured chondrocytes, REDD1 knockdown increased whereas REDD1 overexpression decreased mTOR signaling. In addition, REDD1 activated autophagy by an mTOR independent mechanism that involved protein/protein interaction with TXNIP. The REDD1/TXNIP complex was required for autophagy activation in chondrocytes. CONCLUSION The present study shows that REDD1 is highly expressed in normal human articular cartilage and reduced during aging and OA. REDD1 in human chondrocytes negatively regulates mTOR activity and is essential for autophagy activation. Reduced REDD1 expression thus represents a novel mechanism for the increased mTOR activation and defective autophagy observed in OA.

Transthyretin deposition in articular cartilage: a novel mechanism in the pathogenesis of osteoarthritis.

Amyloid deposits are prevalent in osteoarthritic (OA) joints. We undertook this study to define the dominant precursor and to determine whether the deposits affect chondrocyte functions.

FoxO transcription factors modulate autophagy and proteoglycan 4 in cartilage homeostasis and osteoarthritis

FoxO play a key role in postnatal cartilage development, maturation, homeostasis, and osteoarthritis pathogenesis. Clever as a FoxO FoxO proteins are transcription factors that regulate autophagy, metabolism, and aging. Matsuzaki et al. investigated the role of different FoxO in cartilage development, homeostasis, and degeneration during osteoarthritis. Cartilage was thicker and chondrocytes were more proliferative in young mice lacking FoxO1/3/4 in cartilage. Chondrocyte-specific FoxO-deficient mice exhibited worse arthritis with aging and increased cartilage degradation in response to surgically induced arthritis; they also expressed less lubricin, a protein that helps reduce friction in joints. FoxO1 and autophagy-related genes were reduced in human chondrocytes from patients with osteoarthritis, and restoring FoxO1 expression reduced inflammatory cytokines and up-regulated lubricin. This study suggests that FoxO factors could be targets for therapy in osteoarthritis. Aging is a main risk factor for osteoarthritis (OA). FoxO transcription factors protect against cellular and organismal aging, and FoxO expression in cartilage is reduced with aging and in OA. To investigate the role of FoxO in cartilage, Col2Cre-FoxO1, 3, and 4 single knockout (KO) and triple KO mice (Col2Cre-TKO) were analyzed. Articular cartilage in Col2Cre-TKO and Col2Cre-FoxO1 KO mice was thicker than in control mice at 1 or 2 months of age. This was associated with increased proliferation of chondrocytes of Col2Cre-TKO mice in vivo and in vitro. OA-like changes developed in cartilage, synovium, and subchondral bone between 4 and 6 months of age in Col2Cre-TKO and Col2Cre-FoxO1 KO mice. Col2Cre-FoxO3 and FoxO4 KO mice showed no cartilage abnormalities until 18 months of age when Col2Cre-FoxO3 KO mice had more severe OA than control mice. Autophagy and antioxidant defense genes were reduced in Col2Cre-TKO mice. Deletion of FoxO1/3/4 in mature mice using Aggrecan(Acan)-CreERT2 (AcanCreERT-TKO) also led to spontaneous cartilage degradation and increased OA severity in a surgical model or treadmill running. The superficial zone of knee articular cartilage of Col2Cre-TKO and AcanCreERT-TKO mice exhibited reduced cell density and markedly decreased Prg4. In vitro, ectopic FoxO1 expression increased Prg4 and synergized with transforming growth factor–β stimulation. In OA chondrocytes, overexpression of FoxO1 reduced inflammatory mediators and cartilage-degrading enzymes, increased protective genes, and antagonized interleukin-1β effects. Our observations suggest that FoxO play a key role in postnatal cartilage development, maturation, and homeostasis and protect against OA-associated cartilage damage.

Regulated in Development and DNA Damage Response 1 Deficiency Impairs Autophagy and Mitochondrial Biogenesis in Articular Cartilage and Increases the Severity of Experimental Osteoarthritis

Regulated in development and DNA damage response 1 (REDD1) is an endogenous inhibitor of mechanistic target of rapamycin (mTOR) that regulates cellular stress responses. REDD1 expression is decreased in aged and osteoarthritic (OA) cartilage, and it regulates mTOR signaling and autophagy in articular chondrocytes in vitro. This study was undertaken to investigate the effects of REDD1 deletion in vivo using a mouse model of experimental OA.

REDD1 deficiency impairs autophagy and mitochondrial biogenesis in articular cartilage and increases the severity of experimental osteoarthritis.

Regulated in development and DNA damage response 1 (REDD1) is an endogenous inhibitor of mechanistic target of rapamycin (mTOR) that regulates cellular stress responses. REDD1 expression is decreased in aged and osteoarthritic (OA) cartilage, and it regulates mTOR signaling and autophagy in articular chondrocytes in vitro. This study was undertaken to investigate the effects of REDD1 deletion in vivo using a mouse model of experimental OA.

Increased DNA Methylation and Reduced Expression of Transcription Factors in Human Osteoarthritis Cartilage.

To analyze the methylome of normal and osteoarthritic (OA) knee articular cartilage and to determine the role of DNA methylation in the regulation of gene expression in vitro.

Suppression of Sestrins in aging and osteoarthritic cartilage: dysfunction of an important stress defense mechanism

OBJECTIVES Aging is an important osteoarthritis (OA) risk factor and compromised stress defense responses may mediate this risk. The Sestrins (Sesn) promote cell survival under stress conditions and regulate AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signaling. This study examined Sesn expression in normal and OA cartilage and functions of Sesn in chondrocytes. METHODS Sesn expression in human and mouse normal and OA cartilage was analyzed by quantitative polymerase chain reaction (PCR) and immunohistochemistry. Sesn function was investigated by using small interfering RNA (siRNA) mediated Sesn knockdown and overexpression with analysis of cell survival, gene expression, autophagy, and AMPK and mTOR activation. RESULTS Sesn mRNA levels were significantly reduced in human OA cartilage and immunohistochemistry of human and mouse OA cartilage also showed a corresponding reduction in protein levels. In cultured human chondrocytes Sesn1, 2 and 3 were expressed and increased by tunicamycin, an endoplasmic reticulum (ER) stress response inducer and 2-deoxyglucose (2DG), a metabolic stress inducer. Sesn1 and 2 were increased by tBHP, an oxidative stress inducer. Sesn knockdown by siRNA reduced chondrocyte viability under basal culture conditions and in the presence of 2DG. Sesn overexpression enhanced LC3-II formation and autophagic flux, and this was related to changes in mTOR but not AMPK activation. CONCLUSION These findings are the first to show that Sesn expression is suppressed in OA affected cartilage. Sesn support chondrocyte survival under stress conditions and promote autophagy activation through modulating mTOR activity. Suppression of Sesn in OA cartilage contributes to deficiency in an important cellular homeostasis mechanism.