Exogenous retinoic acid (RA) induces marked effects on limb patterning, but the precise role of endogenous RA in this process has remained unknown. We have studied the role of RA in mouse limb development by focusing on CYP26B1, a cytochrome P450 enzyme that inactivates RA. Cyp26b1 was shown to be expressed in the distal region of the developing limb bud, and mice that lack CYP26B1 exhibited severe limb malformation (meromelia). The lack of CYP26B1 resulted in spreading of the RA signal toward the distal end of the developing limb and induced proximodistal patterning defects characterized by expansion of proximal identity and restriction of distal identity. CYP26B1 deficiency also induced pronounced apoptosis in the developing limb and delayed chondrocyte maturation. Wild-type embryos exposed to excess RA phenocopied the limb defects of Cyp26b1(-/-) mice. These observations suggest that RA acts as a morphogen to determine proximodistal identity, and that CYP26B1 prevents apoptosis and promotes chondrocyte maturation, in the developing limb.

Summary Retinoic acid (RA) is thought to be a key signaling molecule involved in limb bud patterning along the proximodistal or anteroposterior axes functioning through induction of Meis2 and Shh, respectively [1]. Here, we utilize Raldh2-/- and Raldh3-/- mouse embryos lacking RA synthesis [2] to demonstrate that RA signaling is not required for limb expression of Shh and Meis2. We demonstrate that RA action is required outside the limb field in the body axis during forelimb induction, but that RA is unnecessary at later stages when hindlimb budding and patterning occurs. We provide evidence for a model of trunk mesodermal RA action in which forelimb induction requires RA repression of Fgf8 in the developing trunk similar to how RA controls somitogenesis [3, 4] and heart development [5]. We demonstrate that pectoral fin development in RA-deficient zebrafish embryos can be rescued by an FGF receptor antagonist SU5402. In addition, embryo ChIP assays demonstrate that RA receptors bind the Fgf8 promoter in vivo. Our findings suggest that RA signaling is not required for limb proximodistal or anteroposterior patterning but that RA inhibition of FGF8 signaling during the early stages of body axis extension provides an environment permissive for induction of forelimb buds.

IntroductionCD40 is a member of the tumor necrosis factor receptor (TNFR) superfamily that is expressed by cells, including B cells, macrophages, microglia, dendritic cells, endothelial cells, and tumor cells. The interaction between CD40 and its cognate ligand, CD40L (CD154), is critical for a productive immune response. 1,2 Upregulation of various cell surface molecules, such as class II major histocompatibility complex, CD40, CD80, and CD86, occurs on CD40-CD154 contact, as well as the production of numerous cytokines and chemokines (interleukin 1 [IL-1], IL-6, IL-10, tumor necrosis factor ␣ [TNF-␣] and macrophage inflammatory protein 1 [MIP-1]) and cytotoxic radicals. 2 CD40 has been implicated in participating in many human diseases, particularly autoimmune diseases. 3,4 Blocking the interaction between CD40-CD154 with anti-CD154 or anti-CD40 antibody is beneficial in animal models of autoimmune diseases. 5 These findings illustrate the importance of CD40-CD154 interactions for homeostasis of immune responses. We have previously shown that macrophages and microglia, the endogenous macrophage of the brain, constitutively express CD40 at a low level, which is greatly enhanced by the cytokine interferon ␥ (IFN-␥). IFN-␥-induced CD40 expression involves IFN-␥-activated signal transducer and activator of transcription 1␣ (STAT-1␣) and nuclear factor-B (NF-B) activation through an autocrine response to IFN-␥-induced TNF-␣ production. [6][7][8][9] The immune response to microbial pathogens relies on both innate and acquired immunity. Innate immunity is determined by the interaction between potential pathogens and their cognate binding partners (receptors) on phagocytes, which is performed by a group of proteins, the Toll-like receptor (TLR) family. 10,11 At least 10 TLRs (TLR1-TLR10) recognize specific molecular patterns that are present in microbial components. Stimulation of different TLRs induces distinct patterns of gene expression, which not only leads to the activation of innate immunity but also instructs the development of antigen-specific acquired immunity. 12,13 The TLR family signals via shared downstream signaling molecules, including the adapter molecule myeloid differentiation primary-response protein 88 (MyD88), interleukin-1 receptor-associated protein kinases (IRAK1 and IRAK4), transforming growth factor ␤ (TGF-␤)-activated kinase (TAK1), TAK1-binding protein 1 (TAB1), TAB2, and tumor necrosis factor receptor-associated factor 6. 10,11 TAK1 is a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family, which is essential for IL-1, TNF-␣, and lipopolysaccharide (LPS)-induced activation of NF-B and MAPKs. 10 The NF-B family of transcription factors is composed of 5 members, p65 (Rel-A), Rel-B, c-Rel, p50, and p52, which function as homodimers and heterodimers. NF-B transcription factors are present in the cytoplasm in an inactive state, complexed with inhibitory IB proteins. The activation process is mediated by Supported by the National Institutes of Health (grants NS45290 and N...

Objective The etiology of chondrocyte mitochondrial dysfunction in OA is incompletely understood. OA chondrocytes are deficient in active AMPK-activated protein kinase (AMPK) and sirtuin 1 (SIRT1), metabolic biosensors that modulate the mitochondrial biogenesis “master regulator” peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α. Moreover, PGC-1α critically mediates AMPK anti-catabolic activity in chondrocytes. Here, we tested the hypotheses that mitochondrial biogenesis is deficient in human OA chondrocytes, which functionally increases chondrocyte pro-catabolic responses, but is reversed by activation of the AMPK-SIRT1-PGC-1α pathway. Methods We studied human knee chondrocytes, human and mouse knee cartilages. We examined expression and activity (phosphorylation) of AMPKα, and SIRT1 and PGC-1α, and defined and compared mitochondrial content and functions including oxidative phosphorylation (OXPHOS) with expression of mitochondrial biogenesis factors (mitochondrial transcriptional factor A (TFAM), nuclear respiratory factors (NRFs)). Results Human knee OA chondrocytes had decreased mitochondrial biogenesis capacity, linked to reduced AMPKα activity and decreased SIRT1, PGC-1α, TFAM, and NRF1,2 expression. Human knee OA and aged mouse knee cartilages had decreased TFAM and ubiquinol-cytochrome c reductase core protein I (UQCEC1), a subunit of mitochondrial complex III, in situ. Functionally, chondrocyte TFAM knockdown inhibited mitochondrial biogenesis and enhanced pro-catabolic responses to IL-1β. Last, pharmacologic AMPK activation by A-769662 increased PGC-1α via SIRT1, and reversed impairments in mitochondrial biogenesis, OXPHOS, and intracellular ATP in human knee OA chondrocytes. Conclusions Mitochondrial biogenesis is deficient in human OA chondrocytes and this promotes chondrocyte pro-catabolic responses. Activation of the AMPK-SIRT1-PGC-1α pathway reverses these effects, mediated by TFAM, suggesting translational potential to limit OA progression.