Hormone-Independent Sexual Dimorphism in the Regulation of Bone Resorption by Krox20.

Krox20/EGR2 is a zinc finger transcription factor, implicated in the development of the hindbrain, nerve myelination, and tumor suppression. In skeletal biology, we have demonstrated that Krox20 also regulates adult bone metabolism. We and others have characterized several functions of Krox20 in the osteoclast lineage, namely, preosteoclast proliferation and differentiation, and mature osteoclast apoptosis. We have previously reported that systemically Krox20-haploinsufficient mice have a low bone mass with increased bone resorption. However, new data have now revealed that this phenotype is restricted to females. In addition, we discovered that conditional knockout of Krox20 (cKO) restricted to osteoclast progenitors is sufficient to induce the same female-specific bone loss observed in systemic mutants. To test whether this sexual dimorphism results from an interaction between Krox20 and sex hormones, we examined the sex- and hormone-dependent role of Krox20 deficiency on proliferation and apoptosis in osteoclastic cells. Our results indicate that male and female sex hormones (dihydrotestosterone [DHT] and estradiol [E2], respectively) as well as Krox20 inhibit preosteoclast proliferation and augment osteoclast apoptosis. The observation that Krox20 expression is inhibited by DHT and E2 negates the hypothesis that the effect of sex hormones is mediated by an increase in Krox20 expression. Interestingly, the effect of Krox20 deficiency was observed only with cells derived from female animals, regardless of any sex hormones added in vitro. In addition, we have identified sexual dimorphism in the expression of several Krox20-related genes, including NAB2. This sex-specific epigenetic profile was established at puberty, maintained in the absence of sex hormones, and explains the female-specific skeletal importance of Krox20. The findings described in this study emphasize the medical importance of sex differences, which may be determined at the epigenetic level. © 2019 American Society for Bone and Mineral Research.

Egr2 and Egr3 in regulatory T cells cooperatively control systemic autoimmunity through Ltbp3-mediated TGF-ß3 production.

Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease characterized by multiorgan inflammation induced by autoantibodies. Early growth response gene 2 (Egr2), a transcription factor essential for T-cell anergy induction, controls systemic autoimmunity in mice and humans. We have previously identified a subpopulation of CD4+ regulatory T cells, CD4+CD25-LAG3+ cells, that characteristically express both Egr2 and LAG3 and control mice model of lupus via TGF-ß3 production. However, due to the mild phenotype of lymphocyte-specific Egr2-deficient mice, the presence of an additional regulator has been speculated. Here, we show that Egr2 and Egr3 expressed in T cells cooperatively prevent humoral immune responses by supporting TGF-ß3 secretion. T cell-specific Egr2/Egr3 double-deficient (Egr2/3DKO) mice spontaneously developed an early onset lupus-like disease that was more severe than in T cell-specific Egr2-deficient mice. In accordance with the observation that CD4+CD25-LAG3+ cells from Egr2/3DKO mice completely lost the capacity to produce TGF-ß3, the excessive germinal center reaction in Egr2/3DKO mice was suppressed by the adoptive transfer of WT CD4+CD25-LAG3+ cells or treatment with a TGF-ß3-expressing vector. Intriguingly, latent TGF-ß binding protein (Ltbp)3 expression maintained by Egr2 and Egr3 was required for TGF-ß3 production from CD4+CD25-LAG3+ cells. Because Egr2 and Egr3 did not demonstrate cell intrinsic suppression of the development of follicular helper T cells, Egr2- and Egr3-dependent TGF-ß3 production by CD4+CD25-LAG3+ cells is critical for controlling excessive B-cell responses. The unique attributes of Egr2/Egr3 in T cells may provide an opportunity for developing novel therapeutics for autoantibody-mediated diseases including SLE.

Krox20 heterozygous mice: A model of aortic regurgitation associated with decreased expression of fibrillar collagen genes.

BACKGROUND: The mechanism involved in the onset of aortic valve (AoV) disease remains unclear despite its poor prognosis and frequency. Recently, we reported that Krox20 (EGR2 in humans) is involved in AoV development and dysfunction. AIM: Analyze Krox20 heterozygous mice (Krox20(+/-)) to discover whether incomplete expression of Krox20 can cause valvular diseases. METHODS: Transcriptional levels of Col1a2/COL1A2 and Krox20/EGR2 in AoVs from Krox20(+/-) mice and human patients operated on for severe aortic regurgitation were evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Human control valves were obtained from three transplanted patients without AoV disease. Twenty-one heterozygous Krox20(+/-) mice were compared with 35 controls at different ages. Three independent measurements of valve thickness were performed on magnified tissue sections using Image J software. In vivo valve structure and function were evaluated using the high-frequency Vevo(®) 2100 echocardiogram. RESULTS: qRT-PCR analysis using AoVs from patients with severe aortic regurgitation showed a decrease in EGR2 expression associated with significant downregulation of COL1A2 expression (P<0.05). Similar results were observed in the AoVs of Krox20(+/-) mice. Anatomical examination revealed that incomplete invalidation of Krox20 caused significant thickening of the aortic leaflet compared with controls (145±22 vs. 75±24µm; P=0.01). Within the mutant group, this thickening worsened significantly over time (Krox20(+/-) mice aged>7 vs.<7months: 136±48 vs. 102±41µm; P<0.001). Moreover, the aortic leaflets of embryonic day 18.5 Krox20(+/-) embryos were significantly more thickened than those from controls, suggesting that this disease begins during embryonic development. Echo-Doppler analysis showed a significant increase in AoV dysfunction in heterozygous versus control mice (53% vs. 17%; P<0.001), suggesting a tight relationship between valve architecture and function. Morphometric analysis revealed that the most severe AoV dysfunction was always associated with the most thickened valves. Classic histological analysis revealed that mutant AoVs had extracellular matrix disorganization, with features of human myxomatous degeneration, including excess of proteoglycan deposition in spongiosa and reduction of collagen fibre in fibrosa, but no calcification. CONCLUSION: Decreased expression of Krox20 in mice causes degeneration of the aortic leaflets and disorganization of the extracellular matrix, causing valvular dysfunction.

EGR2 is critical for peripheral naïve T-cell differentiation and the T-cell response to influenza.

Early growth response 2 (EGR2) transcription factor negatively regulates T-cell activation, in contrast to the positive regulation of this process by EGR1. Here, we unexpectedly found that EGR2 promotes peripheral naïve T-cell differentiation, with delayed T-cell receptor-induced proliferation in naïve T cells from Egr2 conditional knockout (CKO) mice and decreased production of IFN-γ, IL-4, IL-9, and IL-17A in cells subjected to T-helper differentiation. Moreover, genes that promote T-cell activation, including Tbx21 and Notch1, had decreased expression in Egr2 CKO T cells and are direct EGR2 target genes. Following influenza infection, Egr2 CKO mice had delayed viral clearance, more weight loss, and more severe pathological changes in the lung than did WT and Egr1 KO mice, with decreased production of effector cytokines, increased infiltration of antigen-specific memory-precursor CD8(+) T cells, and lower numbers of lung-resident memory CD8(+) T cells. Thus, unexpectedly, EGR2 can function as a positive regulator that is essential for naïve T-cell differentiation and in vivo T-cell responses to a viral infection.

Egr2-dependent gene expression profiling and ChIP-Seq reveal novel biologic targets in T cell anergy.

T cell anergy is one of the mechanisms contributing to peripheral tolerance, particularly in the context of progressively growing tumors and in tolerogenic treatments promoting allograft acceptance. We recently reported that early growth response gene 2 (Egr2) is a critical transcription factor for the induction of anergy in vitro and in vivo, which was identified based on its ability to regulate the expression of inhibitory signaling molecules diacylglycerol kinase (DGK)-α and -ζ. We reasoned that other transcriptional targets of Egr2 might encode additional factors important for T cell anergy and immune regulation. Thus, we conducted two sets of genome-wide screens: gene expression profiling of wild type versus Egr2-deleted T cells treated under anergizing conditions, and a ChIP-Seq analysis to identify genes that bind Egr2 in anergic cells. Merging of these data sets revealed 49 targets that are directly regulated by Egr2. Among these are inhibitory signaling molecules previously reported to contribute to T cell anergy, but unexpectedly, also cell surface molecules and secreted factors, including lymphocyte-activation gene 3 (Lag3), Class-I-MHC-restricted T cell associated molecule (Crtam), Semaphorin 7A (Sema7A), and chemokine CCL1. These observations suggest that anergic T cells might not simply be functionally inert, and may have additional functional properties oriented towards other cellular components of the immune system.

Early growth response gene-2 controls IL-17 expression and Th17 differentiation by negatively regulating Batf.

Early growth response gene (Egr)-2 is important for the maintenance of T cell homeostasis and controls the development of autoimmune disease. However, the underlying mechanisms are unknown. We have now discovered that Egr-2, which is induced by TGF-ß and IL-6, negatively regulates the expression of IL-17, but not IL-2 or IFN-γ, in effector T cells. In the absence of Egr-2, CD4 T cells produce high levels of Th17 cytokines, which renders mice susceptible to experimental autoimmune encephalomyelitis induction. T cells lacking Egr-2 show increased propensity for Th17, but not Th1 or Th2, differentiation. Control of IL-17 expression and Th17 differentiation by Egr-2 is due to inhibition of Batf, a transcription factor that regulates IL-17 expression and Th17 differentiation. Egr-2 interacts with Batf in CD4 T cells and suppresses its interaction with DNA sequences derived from the IL-17 promoter, whereas the activation of STAT3 and expression of retinoic acid-related orphan receptor γt are unchanged in Th17 cells in the absence of Egr-2. Thus, Egr-2 plays an important role to intrinsically control Th17 differentiation. We also found that CD4 T cells from multiple sclerosis patients have reduced expression of Egr-2 and increased expression of IL-17 following stimulation with anti-CD3 in vitro. Collectively, our results demonstrate that Egr-2 is an intrinsic regulator that controls Th17 differentiation by inhibiting Batf activation, which may be important for the control of multiple sclerosis development.

The transcription factors Egr2 and Egr3 are essential for the control of inflammation and antigen-induced proliferation of B and T cells.

Lymphocytes provide optimal responses against pathogens with minimal inflammatory pathology. However, the intrinsic mechanisms regulating these responses are unknown. Here, we report that deletion of both transcription factors Egr2 and Egr3 in lymphocytes resulted in a lethal autoimmune syndrome with excessive serum proinflammatory cytokines but also impaired antigen receptor-induced proliferation of B and T cells. Egr2- and Egr3-defective B and T cells had hyperactive signal transducer and activator of transcription-1 (STAT1) and STAT3 while antigen receptor-induced activation of transcription factor AP-1 was severely impaired. We discovered that Egr2 and/or Egr3 directly induced expression of suppressor of cytokine signaling-1 (SOCS1) and SOCS3, inhibitors of STAT1 and STAT3, and also blocked the function of Batf, an AP-1 inhibitor, in B and T cells. Thus, Egr2 and Egr3 regulate B and T cell function in adaptive immune responses and homeostasis by promoting antigen receptor signaling and controlling inflammation.

Krox20 inactivation in the PNS leads to CNS/PNS boundary transgression by central glia.

CNS/PNS interfaces constitute cell boundaries, since they delimit territories with different neuronal and glial contents. Despite their potential interest in regenerative medicine, the mechanisms restricting oligodendrocytes and astrocytes to the CNS, and Schwann cells to the PNS in mammals are not known. To investigate the involvement of peripheral glia and myelin in the maintenance of the CNS/PNS boundary, we have first made use of different mouse mutants. We show that inactivation of Krox20/Egr2, a master regulatory gene for myelination in Schwann cells, results in transgression of the CNS/PNS boundary by astrocytes and oligodendrocytes and in myelination of nerve root axons by oligodendrocytes. In contrast, such migration does not occur with the Trembler(J) mutation, which prevents PNS myelination without affecting Krox20 expression. Altogether these data suggest that maintenance of the CNS/PNS boundary requires a new Krox20 function separable from myelination control. Finally, we have analyzed a human patient affected by a congenital amyelinating neuropathy, associated with the absence of the KROX20 protein in Schwann cells. In this case, the nerve roots were also invaded by oligodendrocytes and astrocytes. This indicates that transgression of the CNS/PNS boundary by central glia can occur in pathological situations in humans and suggests that the underlying mechanisms are common with the mouse.

EGR-2 is not required for in vivo CD4 T cell mediated immune responses.

BACKGROUND: The zinc finger transcription factor EGR-2 has been shown to play an important role in the induction of T cell anergy and the regulation of peripheral T cell tolerance. In vitro, a prior study has show that T cells deficient in EGR-2 are hyperproliferative to IL-2 and produce elevated levels of the effector cytokine IFN-γ. EGR-2 deficient mice have increased levels of CD44(high) T cells in peripheral lymphoid organs, and with age, develop autoimmune-like features. PRINCIPAL FINDINGS: Here we show that despite increased numbers of cells bearing an activated CD44(high)CD62L(low) phenotype, T cells from young healthy EGR-2 deficient mice have normal proliferative and cytokine responses, and the mice themselves mount normal immune responses against minor histocompatibility antigens, and the pathogens Toxoplasma gondii and lymphocytic choriomeningitis virus. CONCLUSIONS: Our results indicate that EGR-2 is not required to mount normal acute in vivo immune responses against foreign antigens, and suggest instead that it may serve to regulate the response to chronic antigenic exposure, such as that which occurs to autoantigens.

Krox20/EGR2 deficiency accelerates cell growth and differentiation in the monocytic lineage and decreases bone mass.

Krox20/EGR2, one of the 4 early growth response genes, is a highly conserved transcription factor implicated in hindbrain development, peripheral nerve myelination, tumor suppression, and monocyte/macrophage cell fate determination. Here, we established a novel role for Krox20 in postnatal skeletal metabolism. Microcomputed tomographic analysis of 4- and 8-week-old mice revealed a low bone mass phenotype (LBM) in both the distal femur and the vertebra of Krox20(+/-) mice. This was attributable to accelerated bone resorption as demonstrated in vivo by increased osteoclast number and serum C-terminal telopeptides, a marker for collagen degradation. Krox20 haploinsufficiency did not reduce bone formation in vivo, nor did it compromise osteoblast differentiation in vitro. In contrast, growth and differentiation were significantly stimulated in preosteoclast cultures derived from Krox20(+/-) splenocytes, suggesting that the LBM is attributable to Krox20 haploinsufficiency in the monocytic lineage. Furthermore, Krox20 silencing in preosteoclasts increased cFms expression and response to macrophage colony-stimulating factor, leading to a cell-autonomous stimulation of cell-cycle progression. Our data indicate that the antimitogenic role of Krox20 in preosteoclasts is the predominant mechanism underlying the LBM phenotype of Krox20-deficient mice. Stimulation of Krox20 expression in preosteoclasts may present a viable therapeutic strategy for high-turnover osteoporosis.

Opposing regulation of T cell function by Egr-1/NAB2 and Egr-2/Egr-3.

TCR-induced NF-AT activation leads to the up-regulation of multiple genes involved in T cell anergy. Since NF-AT is also involved in T cell activation, we have endeavored to dissect TCR-induced activating and inhibitory genetic programs. This approach revealed roles for the early growth response (Egr) family of transcription factors and the Egr coactivator/corepressor NGFI-A-binding protein (NAB)2 in regulating T cell function. TCR-induced Egr-1 and NAB2 enhance T cell function, while Egr-2 and Egr-3 inhibit T cell function. In this report, we demonstrate that Egr-2 and Egr-3 are induced by NF-AT in the absence of AP-1, while Egr-1 and NAB2 both require AP-1-mediated transcription. Our data suggest that Egr-3 is upstream of Egr-2, and that mechanistically Egr-2 and Egr-3 suppress Egr-1 and NAB2 expression. Functionally, T cells from Egr-2 and Egr-3 null mice are hyperresponsive while T cells from Egr-3 transgenic, overexpressing mice are hyporesponsive. Furthermore, an in vivo model of autoimmune pneumonitis reveals that T cells from Egr-3 null mice hasten death while Egr-3-overexpressing T cells cause less disease. Overall, our data suggest that just as the Egr/NAB network of genes control cell fate in other systems, TCR-induced Egr-1, 2, 3 and NAB2 control the fate of antigen recognition in T cells.

Krox-20 gene expression: influencing hindbrain-craniofacial developmental interactions.

Krox-20 is a C(2)H(2)-type zinc-finger transcription factor that plays an essential role in hindbrain development. The Krox-20 null mutation results in hindbrain anomalies that result in neonatal death due to respiratory and feeding deficits. Here we review our studies of how the Krox- 20 null mutation impacts the development of motor and sensory systems critical for the production of consummatory behaviors (suckling/chewing). First, we demonstrated that Krox-20 null mutants suffer a selective loss of primary jaw-opening muscles during prenatal development. In vivo and in vitro studies are reviewed that highlight intrinsic defects in mutant jaw-opener muscles that contribute to muscle degeneration. Next we focus on the impact of the mutation on proprioceptive neurons activated during consummatory behaviors. Mesencephalic trigeminal (Me5) neurons are primary sensory neurons that relay jaw proprioception to the central nervous system. These cells are unique because their cell bodies are located in the central as opposed to the peripheral nervous system. Data are reviewed that demonstrate the impact of the mutation on Me5 neurons, a cell group traditionally thought to emerge from the mesencephalon. We show that Krox-20 null mutants have twice as many Me5 neurons relative to wildtypes at E15, but by birth have half the number of Me5 cells as wildtypes. TUNEL assays performed in each set of studies reveal that Krox-20 expression acts to protect both muscle and mesencephalic trigeminal neurons against apoptosis, suggesting that Krox-20, in addition to its role in hindbrain patterning, has a broader, long-lasting role in development.