Control of mitochondrial function and cell growth by the atypical cadherin Fat1.

Mitochondrial products such as ATP, reactive oxygen species, and aspartate are key regulators of cellular metabolism and growth. Abnormal mitochondrial function compromises integrated growth-related processes such as development and tissue repair, as well as homeostatic mechanisms that counteract ageing and neurodegeneration, cardiovascular disease, and cancer. Physiologic mechanisms that control mitochondrial activity in such settings remain incompletely understood. Here we show that the atypical Fat1 cadherin acts as a molecular 'brake' on mitochondrial respiration that regulates vascular smooth muscle cell (SMC) proliferation after arterial injury. Fragments of Fat1 accumulate in SMC mitochondria, and the Fat1 intracellular domain interacts with multiple mitochondrial proteins, including critical factors associated with the inner mitochondrial membrane. SMCs lacking Fat1 (Fat1KO) grow faster, consume more oxygen for ATP production, and contain more aspartate. Notably, expression in Fat1KO cells of a modified Fat1 intracellular domain that localizes exclusively to mitochondria largely normalizes oxygen consumption, and the growth advantage of these cells can be suppressed by inhibition of mitochondrial respiration, which suggest that a Fat1-mediated growth control mechanism is intrinsic to mitochondria. Consistent with this idea, Fat1 species associate with multiple respiratory complexes, and Fat1 deletion both increases the activity of complexes I and II and promotes the formation of complex-I-containing supercomplexes. In vivo, Fat1 is expressed in injured human and mouse arteries, and inactivation of SMC Fat1 in mice potentiates the response to vascular damage, with markedly increased medial hyperplasia and neointimal growth, and evidence of higher SMC mitochondrial respiration. These studies suggest that Fat1 controls mitochondrial activity to restrain cell growth during the reparative, proliferative state induced by vascular injury. Given recent reports linking Fat1 to cancer, abnormal kidney and muscle development, and neuropsychiatric disease, this Fat1 function may have importance in other settings of altered cell growth and metabolism.

Inhibition of Smooth Muscle ß-Catenin Hinders Neointima Formation After Vascular Injury.

OBJECTIVE: Smooth muscle cells (SMCs) contribute to neointima formation after vascular injury. Although ß-catenin expression is induced after injury, whether its function is essential in SMCs for neointimal growth is unknown. Moreover, although inhibitors of ß-catenin have been developed, their effects on SMC growth have not been tested. We assessed the requirement for SMC ß-catenin in short-term vascular homeostasis and in response to arterial injury and investigated the effects of ß-catenin inhibitors on vascular SMC growth. APPROACH AND RESULTS: We used an inducible, conditional genetic deletion of ß-catenin in SMCs of adult mice. Uninjured arteries from adult mice lacking SMC ß-catenin were indistinguishable from controls in terms of structure and SMC marker gene expression. After carotid artery ligation, however, vessels from mice lacking SMC ß-catenin developed smaller neointimas, with lower neointimal cell proliferation and increased apoptosis. SMCs lacking ß-catenin showed decreased mRNA expression of Mmp2, Mmp9, Sphk1, and S1pr1 (genes that promote neointima formation), higher levels of Jag1 and Gja1 (genes that inhibit neointima formation), decreased Mmp2 protein expression and secretion, and reduced cell invasion in vitro. Moreover, ß-catenin inhibitors PKF118-310 and ICG-001 limited growth of mouse and human vascular SMCs in a dose-dependent manner. CONCLUSIONS: SMC ß-catenin is dispensable for maintenance of the structure and state of differentiation of uninjured adult arteries, but is required for neointima formation after vascular injury. Pharmacological ß-catenin inhibitors hinder growth of human vascular SMCs. Thus, inhibiting ß-catenin has potential as a therapy to limit SMC accumulation and vascular obstruction.

Loss of Allograft Inflammatory Factor-1 Ameliorates Experimental Autoimmune Encephalomyelitis by Limiting Encephalitogenic CD4 T-Cell Expansion.

Experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis (MS), is mediated by myelin-specific autoreactive T cells that cause inflammation and demyelination in the central nervous system (CNS), with significant contributions from activated microglia and macrophages. The molecular bases for expansion and activation of these cells, plus trafficking to the CNS for peripheral cells, are not fully understood. Allograft inflammatory factor-1 (Aif-1) (also known as ionized Ca(2+) binding adapter-1 [Iba-1]) is induced in leukocytes in MS and EAE; here we provide the first assessment of Aif-1 function in this setting. After myelin oligodendrocyte glycoprotein peptide (MOG35-55) immunization, Aif-1-deficient mice were less likely than controls to develop EAE and had less CNS leukocyte infiltration and demyelination; their spinal cords contained fewer CD4 T cells and microglia and more CD8 T cells. These mice also showed significantly less splenic CD4 T-cell expansion and activation, plus decreased proinflammatory cytokine expression. These findings identify Aif-1 as a potent molecule that promotes expansion and activation of CD4 T cells, plus elaboration of a proinflammatory cytokine milieu, in MOG35-55-induced EAE and as a potential therapeutic target in MS.

Donor and recipient cell surface colony stimulating factor-1 promote neointimal formation in transplant-associated arteriosclerosis.

OBJECTIVE: Transplant-associated arteriosclerosis manifests as progressive vascular neointimal expansion throughout the arterial system of allografted solid organs, and eventually compromises graft perfusion and function. Allografts placed in colony stimulating factor (CSF)-1-deficient osteopetrotic (Csf1(op)/Csf1(op)) mice develop very little neointima, a finding attributed to impaired recipient macrophage function. We examined how CSF-1 affects neointima-derived vascular smooth muscle cells, tested the significance of CSF-1 expressed in donor tissue, and evaluated the contribution of secreted versus cell surface CSF-1 isoforms in transplant-associated arteriosclerosis. METHODS AND RESULTS: CSF-1 activated specific signaling pathways to promote migration, survival, and proliferation of cultured vascular smooth muscle cells. Tumor necrosis factor-α addition increased CSF-1 and CSF-1 receptor expression, and tumor necrosis factor-α-driven proliferation was blocked by anti-CSF-1 antibody. In a mouse vascular allograft model, lack of recipient or donor CSF-1 impaired neointima formation; the latter suggests local CSF-1 function within the allograft. Moreover, reconstitution of donor or recipient cell surface CSF-1, without secreted CSF-1, restored neointimal formation. CONCLUSIONS: Vascular smooth muscle cells activation, including that mediated by tumor necrosis factor-α, can be driven in an autocrine/juxtacrine manner by CSF-1. These studies provide evidence for local function of CSF-1 in neointimal expansion, and identify CSF-1 signaling in vascular smooth muscle cells, particularly cell surface CSF-1 signaling, as a target for therapeutic strategies in transplant-associated arteriosclerosis.

Genetic inactivation of the allograft inflammatory factor-1 locus.

Allograft inflammatory factor-1 (Aif-1) is a 17 kDa EF hand motif-bearing protein expressed primarily in developing spermatids and cells of monocyte/macrophage lineage. Increased Aif-1 expression has been identified in clinically important conditions, including rheumatoid arthritis, systemic sclerosis, endometriosis, and transplant-associated arteriosclerosis. Largely similar gene products arising from the same locus are known as ionized Ca(2+) binding adapter-1 (Iba1), microglial response factor-1 (MRF1), and daintain; Iba1 in particular has emerged as a histologic marker of microglia and their activation in pathologic CNS conditions, including the response to facial nerve axotomy and stroke, uveitis, and experimental autoimmune neuritis and encephalomyelitis. Nevertheless, how aif-1 gene products affect cellular function is only partly understood, and the physiologic significance of these products for male fertility, immune system development, and inflammation has not been described. To permit such investigations, we generated a mouse line with targeted deletion of the coding regions of the aif-1 gene. Here we report that mice lacking Aif-1 breed well and show normal post-natal growth, but show resistance to disease in a model of collagen-induced arthritis. We anticipate that these mice will be useful for studies of Aif-1 function in a variety of immune and inflammatory disease models.

Antigen is required for maturation and activation of pathogenic anti-DNA antibodies and systemic inflammation.

Systemic lupus erythematosus is an autoimmune disease characterized by autoantibodies and systemic inflammation that results in part from dendritic cell activation by nucleic acid containing immune complexes. There are many mouse models of lupus, some spontaneous and some induced. We have been interested in an induced model in which estrogen is the trigger for development of a lupus-like serology. The R4A transgenic mouse expresses a transgene-encoded H chain of an anti-DNA Ab. This mouse maintains normal B cell tolerance with deletion of high-affinity DNA-reactive B cells and maturation to immunocompetence of B cells making nonglomerulotropic, low-affinity DNA-reactive Abs. When this mouse is given estradiol, normal tolerance mechanisms are altered; high-affinity DNA-reactive B cells mature to a marginal zone phenotype, and the mice are induced to make high titers of anti-DNA Abs. We now show that estradiol administration also leads to systemic inflammation with increased B cell-activating factor and IFN levels and induction of an IFN signature. DNA must be accessible to B cells for both the production of high-affinity anti-DNA Abs and the generation of the proinflammatory milieu. When DNase is delivered to the mice at the same time as estradiol, there is no evidence for an abrogation of tolerance, no increased B cell-activating factor and IFN, and no IFN signature. Thus, the presence of autoantigen is required for positive selection of autoreactive B cells and for the subsequent positive feedback loop that occurs secondary to dendritic cell activation by DNA-containing immune complexes.

Increased adipose catecholamine levels and protection from obesity with loss of Allograft Inflammatory Factor-1.

Recent studies implicate macrophages in regulation of thermogenic, sympathetic neuron-mediated norepinephrine (NE) signaling in adipose tissues, but understanding of such non-classical macrophage activities is incomplete. Here we show that male mice lacking the allograft inflammatory factor-1 (AIF1) protein resist high fat diet (HFD)-induced obesity and hyperglycemia. We link this phenotype to higher adipose NE levels that stem from decreased monoamine oxidase A (MAOA) expression and NE clearance by AIF1-deficient macrophages, and find through reciprocal bone marrow transplantation that donor Aif1-/- vs WT genotype confers the obesity phenotype in mice. Interestingly, human sequence variants near the AIF1 locus associate with obesity and diabetes; in adipose samples from participants with obesity, we observe direct correlation of AIF1 and MAOA transcript levels. These findings identify AIF1 as a regulator of MAOA expression in macrophages and catecholamine activity in adipose tissues - limiting energy expenditure and promoting energy storage - and suggest how it might contribute to human obesity.

Differential roles of estrogen receptors α and ß in control of B-cell maturation and selection.

It is clear that estrogen can accelerate and exacerbate disease in some lupus-prone mouse strains. It also appears that estrogen can contribute to disease onset or flare in a subset of patients with lupus. We have previously shown estrogen alters B-cell development to decrease lymphopoiesis and increase the frequency of marginal zone B cells. Furthermore, estrogen diminishes B-cell receptor signaling and allows for the increased survival of high-affinity DNA-reactive B cells. Here, we analyze the contribution of estrogen receptor α or ß engagement to the altered B-cell maturation and selection mediated by increased exposure to estrogen. We demonstrate that engagement of either estrogen receptor α or ß can alter B-cell maturation, but only engagement of estrogen receptor α is a trigger for autoimmunity. Thus, maturation and selection are regulated differentially by estrogen. These observations have therapeutic implications.

Induction of interferon signaling and allograft inflammatory factor 1 in macrophages in a mouse model of breast cancer metastases.

Background: Metastatic breast cancer cells recruit macrophages (metastasis-associated macrophages, or MAMs) to facilitate their seeding, survival and outgrowth. However, a comprehensive understanding of the gene expression program in MAMs and how this program contributes to metastasis remain elusive. Methods: We compared the transcriptomes of MAMs recruited to lung metastases and resident alveolar macrophages (RAMs) and identified a large variety of differentially expressed genes and their associated signaling pathways. Some of the changes were validated using qRT-PCR and immunofluorescence. To probe the functional relevance to metastatic growth, a gene-targeting mouse model of female mice in the C57BL6/J background was used to study allograft inflammatory factor 1 (AIF1, also known as ionized calcium-binding adapter molecule 1 or IBA1). Results: Interferon signaling is one of the most activated pathways in MAMs, with strong upregulation of multiple components of the pathway and a significant enrichment for the gene signatures of interferon-alpha-treated human macrophages. Aif1, an interferon-responsive gene that regulates multiple macrophage activities, was robustly induced in MAMs. Aif1 deficiency in MAMs, however, did not affect development of lung metastases, suggesting that AIF1 indicates MAM activation but is dispensable for regulating metastasis. Conclusions: The drastically different gene expression profile of MAMs as compared to RAMs suggests an important role in promoting metastatic growth. Dissection of the underlying mechanisms and functional validation of potential targets in the profile may provide novel therapeutic strategies for the treatment of metastatic diseases.

Allograft inflammatory factor-1-like is not essential for age dependent weight gain or HFD-induced obesity and glucose insensitivity.

The allograft inflammatory factor (AIF) gene family consists of two identified paralogs - AIF1 and AIF1-like (AIF1L). The encoded proteins, AIF1 and AIF1L, are 80% similar in sequence and show conserved tertiary structure. While studies in human populations suggest links between AIF1 and metabolic diseases such as obesity and diabetes, such associations with AIF1L have not been reported. Drawing parallels based on structural similarity, we postulated that AIF1L might contribute to metabolic disorders, and studied it using mouse models. Here we report that AIF1L is expressed in major adipose depots and kidney but was not detectable in liver or skeletal muscle; in notable contrast to AIF1, AIF1L was also not found in spleen. Studies of AIF1L deficient mice showed no obvious postnatal developmental phenotype. In response to high fat diet (HFD) feeding for 6 or 18 weeks, WT and AIF1L deficient mice gained weight similarly, showed no differences in fat or lean mass accumulation, and displayed no changes in energy expenditure or systemic glucose handling. These findings indicate that AIF1L is not essential for the development of obesity or impaired glucose handling due to HFD, and advance understanding of this little-studied gene and its place in the AIF gene family.

Selective regulation of autoreactive B cells by FcgammaRIIB.

FcgammaRIIB is an inhibitory receptor which plays a role in limiting B cell and DC activation. Since FcgammaRIIB is known to dampen the signaling strength of the BCR, we wished to determine the impact of FcgammaRIIB on the regulation of BCRs which differ in their affinity for DNA. For these studies, FcgammaRIIB deficient BALB/c mice were bred with mice expressing the transgene-encoded H chain of the R4A anti-DNA antibody which gives rise to BCRs which express high, low or no affinity for DNA. The deletion of FcgammaRIIB in R4A BALB/c mice led to an alteration in the B cell repertoire, allowing for the expansion and activation of high affinity DNA-reactive B cells. By 6-8 months of age, R4A x FcgammaRIIB-/- BALB/c mice spontaneously developed anti-DNA antibody titers. These mice also displayed an induction of IFN-inducible genes and an elevation in levels of the B cell survival factor, BAFF. These data demonstrate that FcgammaRIIB preferentially limits activation of high affinity autoreactive B cells and can influence the activation of DC through an immune complex-mediated mechanism.

Allograft inflammatory factor-1 supports macrophage survival and efferocytosis and limits necrosis in atherosclerotic plaques.

BACKGROUND AND AIMS: Allograft inflammatory factor-1 (AIF1) has been characterized as a pro-inflammatory molecule expressed primarily in the monocyte/macrophage (MP) lineage and positively associated with various forms of vascular disease, including atherosclerosis. Studies of AIF1 in atherosclerosis have relied on mouse models in which AIF1 was overexpressed in either myeloid or smooth muscle cells, resulting in increased atherosclerotic plaque burden. How physiologic expression of AIF1 contributes to MP biology in atherogenesis is not known. METHODS: Effects of global AIF1 deficiency on atherosclerosis were assessed by crossing Aif1-/- and ApoE-/- mice, and provoking hyperlipidemia with high fat diet feeding. Atherosclerotic plaques were studied en face and in cross section. Bone marrow-derived MPs (BMDMs) were isolated from Aif1-/- mice for study in culture. RESULTS: Atherosclerotic plaques in Aif1-/-;ApoE-/- mice showed larger necrotic cores compared to those in ApoE-/- animals, without change in overall lesion burden. In vitro, lack of AIF1 reduced BMDM survival, phagocytosis, and efferocytosis. Mechanistically, AIF1 supported activation of the NF-κB pathway and expression of related target genes involved in stress response, inflammation, and apoptosis. Consistent with this in vitro BMDM phenotype, AIF1 deficiency reduced NF-κB pathway activity in vivo and increased apoptotic cell number in atherosclerotic lesions from Aif1-/-;ApoE-/- mice. CONCLUSIONS: These findings characterize AIF1 as a positive regulator of the NF-κB pathway that supports MP functions such as survival and efferocytosis. In inflammatory settings such as atherosclerosis, these AIF1-dependent activities serve to clear cellular and other debris and limit necrotic core expansion, and may oppose lesion destabilization.

Identification of DNA-reactive B cells in patients with systemic lupus erythematosus.

Autoreactive B cells play a central role in systemic lupus erythematosus (SLE). Characterization of DNA-reactive B cells in the blood of lupus patients has been limited by the low frequency of the population. Using a tetrameric configuration of a peptide mimetope of DNA, we identified peptide-reactive B cells in peripheral blood. Antibodies derived from these B cells bound to peptide and were largely cross-reactive to dsDNA. This methodology enables us to track the development of autoreactive B cells, which recognize peptide and dsDNA, in individual patients with SLE and permits the isolation of autoreactive B cells for further characterization.

ß-Catenin C-terminal signals suppress p53 and are essential for artery formation.

Increased activity of the tumour suppressor p53 is incompatible with embryogenesis, but how p53 is controlled is not fully understood. Differential requirements for p53 inhibitors Mdm2 and Mdm4 during development suggest that these control mechanisms are context-dependent. Artery formation requires investment of nascent endothelial tubes by smooth muscle cells (SMCs). Here, we find that embryos lacking SMC ß-catenin suffer impaired arterial maturation and die by E12.5, with increased vascular wall p53 activity. ß-Catenin-deficient SMCs show no change in p53 levels, but greater p53 acetylation and activity, plus impaired growth and survival. In vivo, SMC p53 inactivation suppresses phenotypes caused by loss of ß-catenin. Mechanistically, ß-catenin C-terminal interactions inhibit Creb-binding protein-dependent p53 acetylation and p53 transcriptional activity, and are required for artery formation. Thus in SMCs, the ß-catenin C-terminus indirectly represses p53, and this function is essential for embryogenesis. These findings have implications for angiogenesis, tissue engineering and vascular disease.