Mesenchymal responses are an essential aspect of tissue repair. Failure to terminate this repair process correctly, however, results in fibrosis and organ dysfunction. Therapies that block fibrosis and restore tissue homeostasis are not yet available for clinical use. Here we characterize the nuclear receptor NR4A1 as an endogenous inhibitor of transforming growth factor-β (TGF-β) signaling and as a potential target for anti-fibrotic therapies. NR4A1 recruits a repressor complex comprising SP1, SIN3A, CoREST, LSD1, and HDAC1 to TGF-β target genes, thereby limiting pro-fibrotic TGF-β effects. Even though temporary upregulation of TGF-β in physiologic wound healing induces NR4A1 expression and thereby creates a negative feedback loop, the persistent activation of TGF-β signaling in fibrotic diseases uses AKT- and HDAC-dependent mechanisms to inhibit NR4A1 expression and activation. Small-molecule NR4A1 agonists can overcome this lack of active NR4A1 and inhibit experimentally-induced skin, lung, liver, and kidney fibrosis in mice. Our data demonstrate a regulatory role of NR4A1 in TGF-β signaling and fibrosis, providing the first proof of concept for targeting NR4A1 in fibrotic diseases.

Objectives Autophagy is a homeostatic process to recycle dispensable and damaged cell organelles. Dysregulation of autophagic pathways has recently been implicated in the pathogenesis of various diseases. Here, we investigated the role of autophagy during joint destruction in arthritis. Methods Autophagy in osteoclasts was analysed in vitro and ex vivo by transmission electron microscopy, Western blotting and immunohistochemistry for Beclin1 and Atg7. Small molecule inhibitors, LysMCre-mediated knockout of Atg7 and lentiviral overexpression of Beclin1 were used to modulate autophagy in vitro and in vivo. Osteoclast differentiation markers were quantified by realtime PCR. The extent of bone and cartilage destruction was analysed in human tumour necrosis factor α transgenic (hTNFα tg) mice after adoptive transfer with myeloid specific Atg7-deficient bone marrow. Results Autophagy was activated in osteoclasts of human rheumatoid arthritis (RA) showing increased expression of Beclin1 and Atg7. TNFα potently induced the expression of autophagy-related genes and activated autophagy in vitro and in vivo. Activation of autophagy by overexpression of Beclin1-induced osteoclastogenesis and enhanced the resorptive capacity of cultured osteoclasts, whereas pharmacologic or genetic inactivation of autophagy prevented osteoclast differentiation. Arthritic hTNFα tg mice transplanted with Atg7 fl/fl ×LysMCre + bone marrow cells (BMC) showed reduced numbers of osteoclasts and were protected from TNFα-induced bone erosion, proteoglycan loss and chondrocyte death. Conclusions These findings demonstrate that autophagy is activated in RA in a TNFα-dependent manner and regulates osteoclast differentiation and bone resorption. We thus provide evidence for a central role of autophagy in joint destruction in RA.

IL-33 is a new member of the IL-1 family, which plays a crucial role in inflammatory response, enhancing the differentiation of dendritic cells and alternatively activated macrophages (AAM). Based on the evidence of IL-33 expression in bone, we hypothesized that IL-33 may shift the balance from osteoclast to AAM differentiation and protect from inflammatory bone loss. Using transgenic mice overexpressing human TNF, which develop spontaneous joint inflammation and cartilage destruction, we show that administration of IL-33 or an IL-33R (ST2L) agonistic Ab inhibited cartilage destruction, systemic bone loss, and osteoclast differentiation. Reconstitution of irradiated hTNFtg mice with ST2−/− bone marrow led to more bone loss compared with the chimeras with ST2+/+ bone marrow, demonstrating an important endogenous role of the IL-33/ST2L pathway in bone turnover. The protective effect of IL-33 on bone was accompanied by a significant increase of antiosteoclastogenic cytokines (GM-CSF, IL-4, and IFN-γ) in the serum. In vitro IL-33 directly inhibits mouse and human M-CSF/receptor activator for NF-κB ligand-driven osteoclast differentiation. IL-33 acts directly on murine osteoclast precursors, shifting their differentiation toward CD206+ AAMs via GM-CSF in an autocrine fashion. Thus, we show in this study that IL-33 is an important bone-protecting cytokine and may be of therapeutic benefit in treating bone resorption.

Peroxisome proliferator-activated receptors (PPARs) act as metabolic sensors and central regulators of fat and glucose homeostasis. Furthermore, PPARγ has been implicated as major catabolic regulator of bone mass in mice and humans. However, a potential involvement of other PPAR subtypes in the regulation of bone homeostasis has remained elusive. Here we report a previously unrecognized role of PPARβ/δ as a key regulator of bone turnover and the crosstalk between osteoblasts and osteoclasts. In contrast to activation of PPARγ, activation of PPARβ/δ amplified Wnt-dependent and β-catenin-dependent signaling and gene expression in osteoblasts, resulting in increased expression of osteoprotegerin (OPG) and attenuation of osteoblast-mediated osteoclastogenesis. Accordingly, PPARβ/δ-deficient mice had lower Wnt signaling activity, lower serum concentrations of OPG, higher numbers of osteoclasts and osteopenia. Pharmacological activation of PPARβ/δ in a mouse model of postmenopausal osteoporosis led to normalization of the altered ratio of tumor necrosis factor superfamily, member 11 (RANKL, also called TNFSF11) to OPG, a rebalancing of bone turnover and the restoration of normal bone density. Our findings identify PPARβ/δ as a promising target for an alternative approach in the treatment of osteoporosis and related diseases.