Abstract Introduction The aryl hydrocarbon receptor (AHR) integrates metabolic and immune cues, but its role in B-cell regulation during lupus remains unclear. We investigated whether AHR restrains pathogenic B-cell development under lupus-mimicking conditions using toll-like receptor 7 (TLR7) stimulation. Methods B-cell-specific Ahr knockout (Ahrf/f Cd19.Cre+/- or Ahr B-KO) mice were generated on B6 and lupus-prone BXD2 backgrounds and compared with Ahr B-wild type (Ahr B-WT) controls. TLR7 was activated using R848. AHR was activated using tryptophan (Trp) metabolites 6-formylindolo[3,2-b]carbazole (FICZ) and kynurenine (KYN). B cell subpopulations were determined by flow cytometry. In vitro B cell responses to FICZ were analyzed for transcriptomics using the 10x Chromium single cell RNA-sequencing (scRNA-seq) method while the metabolomics of B cells was analyzed by untargeted LC-MS/MS. Results In vivo, FICZ reduced R848-induced T-bet+CXCR3+ B cells, germinal center B cells, and CXCR3+CD138+ plasma cells in BXD2 mice. Loss of Ahr in B cells resulted in an increase in R848-induced anti-DNA IgM⁻IgD⁻ and human DN2-equivalents (T-bet+FCRL5+, T-bet+CXCR3+) B cells in B6 Ahr B-KO, compared to Ahr B-WT mice. In vitro, AHR activation preserved naïve B cells and blocked their conversion into T-bet+CD11c+CXCR3+ cells, but this protection was absent in BXD2 Ahr B-KO B cells. AHR activation increased KYN- and indole-pyruvic acid (IPyA) derived AHR agonistic ligand metabolites in BXD2 Ahr intact but not Ahr deficient B cells. scRNA-seq confirmed upregulation of IPyA (Il4i1) and KYN (Kynu, Kmo, Haao) catabolic enzyme genes in BXD2 Ahr B-WT but not Ahr B-KO B cells. Conclusion These findings identify AHR as a metabolic-immune checkpoint that restrains lupus-related autoantibody production. By uncovering a B-cell intrinsic self-reinforcing pathway that links Trp metabolism to AHR signaling, this work reveals AHR as a promising therapeutic target in lupus and other autoimmune diseases. Funding Source VA Merit Review grant (1I01BX006099), NIH R01 AI134023, and 1R01AI182085 to J.D.M. Topic Categories Therapeutic Approaches to Autoimmunity (THER)

TLR7 stimulation of T-bet+CD11c+IgD-CD27- double-negative 2 (DN2) B cells is crucial for autoantibody formation in systemic lupus erythematosus (SLE). Here, we show that administration of IL-4 for five weeks significantly reduced autoantibodies and T-bet+CD11c+ IgD- B cells in autoimmune BXD2 mice treated with R848, a TLR7 agonist. Single-cell transcriptomics analysis indicates that following two doses of in vivo administration, IL-4 redirected development toward follicular, CD23+ germinal center (GC), and DN4-like memory B cells compared to treatment with R848 alone. While IL-4 enhanced genes related to antigen processing and presentation, it also suppressed R848-induced Ki67+ GC B cells in vivo. In vitro stimulation of SLE patient B cells with a DN2 polarizing cocktail revealed that IL-4 reduced the expression of interferon response and DN2 signature genes, promoting a population of CD23+T-bet- DN4 B population. These findings suggest that developmental reprogramming by IL-4 counteracts TLR7-promoted DN2 and GC B cells in SLE.

Systemic lupus erythematosus (SLE) is marked by an increase in T-bet+CXCR3+Fcrl5+IgD−CD27− double negative 2 (DN2) B cells, driven by heightened TLR7 signaling. We found that SLE patients have reduced aryl hydrocarbon receptor (AhR) expression in naïve and DN B cells compared to healthy controls, along with lower expression of the AhR target gene, Cyp1a1. To investigate the role of AhR in B cell differentiation, we studied B cells from B-cell-specific AhR knockout mice (AhRf/f x Cd19.Crehet: AhR B-KO) and wild-type (AhR+/+ x Cd19.Crehet: AhR B-WT) controls. In vivo administration of mice with R848, a TLR7 ligand, showed an increase in Fcrl5+T-bet+ B cells in both the follicular (p = 0.018) and marginal zone (p = 0.014) subsets in AhR B-KO, compared to AhR B-WT mice. AhR deficiency in B cells also led to a rise in CD80+CXCR3+ populations at IgD + (p = 0.005) and IgD−IgM − (p = 0.023) stages. In vivo treatment of R848-injected BXD2 mice with kynurenine (Kyn), a natural agonistic ligand of AhR, significantly reduced CD11c+Ki67+CD138+ plasma B cells (p = 0.0112) and CD11c+T-bet+ B cells in the germinal center (p = 0.025). These findings suggest that impaired AhR signaling may drive abnormal activation and DN2 B cell development in SLE. In addition, our results highlight the potential of activating AhR with natural ligands such as Kyn to inhibit autoreactive B cell development in SLE. VA Merit Review grant (1I01BX006099) and NIH R01 AI134023 to J.D.M. Basic Autoimmunity (BA)

Background/Purpose We recently showed that in SLE, IL-4 suppressed the development of interferon-beta (IFNβ) and TLR7-stimulated T-bet+ double negative 2 (DN2) B cells. Here we investigate the potential DN2 B-cell suppressive effects of IL-4 through the IL- 4-induced 1 (IL-4i1)-aryl-hydrocarbon receptor (AhR) pathway. Methods The mechanism of IL-4 in suppressing the development of DN2 B cells in vivo was studied using R848-treated BXD2 mice. The B-cell developmental trajectory was determined using single-cell RNA-sequencing (scRNA-seq) analysis. Adult patients who met the ACR 1997 revised criteria for SLE were recruited. The in vitro effects of IL- 4 and two potent AhR agonistic ligands, Kynurenine (Kyn) and 6-formylindolo[3,2- b]carbazole (FICZ) in suppressing the development of DN2 B cell were determined using IFNβ plus TLR7 stimulated B cells. B-cell subsets and transcription factors (TFs) expression was measured by surface and intra-nuclear FACS analysis. AhR pathway and target genes were analyzed using qPCR. Autoantibodies were measured by ELISA. Results Administration of IL-4 significantly inhibited the development of anti-Smith, anti-DNA, and anti-histone autoantibodies induced by the TLR7 agonist R848 in BXD2 mice. This was associated with a decreased percentage of CD11c+T-bet+ IgD− B cells. Feature-barcoding single-cell RNA-sequencing analysis showed that IL-4 modulated B- cell development at the transitional stage 2 (T2) and skewed naïve B cells to develop into the CD23+CD21− follicular B cells. IL-4 induced the gene encoding Ii4i1, an enzyme that metabolizes aromatic amino acids, and this was associated with the upregulation of AhR and downstream genes Cyp1a1 and Ido1. In the absence of IL-4, both Kyn and FICZ significantly suppressed TLR7 plus IFNβ-induced T-bet+ B-cell development in vitro in BXD2 mice. Analysis of AhR expression in healthy control (HC) and SLE subjects show a significantly lower expression of AhR in both the IgD−CD27− DN and IgD+CD27− naïve B-cell populations of SLE compared to HC. qPCR analysis further shows a significantly lower expression of CYP1A1 and IL4I1 in SLE B cells compared to HC B cells. B cell culture with either IL-4 or Kyn significantly reduced the development of DN2 B cells stimulated by IFNβ plus TLR7. Kyn also significantly induced the expression of CYP1A1 and promoted the expression of PD-1 in IFNβ plus TLR7- stimulated B cells. Conclusion Our results suggest that IL-4R acts through the IL4i1-AhR pathway to induce a B-cell regulatory response to TLR7 and type I IFN. Identifying small molecular metabolites that act directly in B cells to induce homeostasis may lead to the development of orally active druggable targets that are efficacious in treating SLE. This study was supported by a VA Merit Review grant (I01BX004049 and I01BX006099), NIH grants R01 AI134023, a Lupus Research Alliance Distinguished Innovator Award to JDM, an LRA Target Identification in Lupus Award to H-C.H., and the P30-AR-048311

Figure S1: (Overexpression of CD24 mRNA in human PCa tissue based on analysis of three datasets); Figure S2: (Nuclear CD24 and p53 staining in human PCa cases); Figure S3: (The sequence-specific DNA binding sites of p53 in its target genes); Figure S4: (CD24-dependent inactivation of mutant p53 and cell cycle arrast in human LNCaP cells).

Cardinal features of lupus include elevated B cell activation and autoantibody production with a female sex preponderance. We quantified interactions of sex and genetic variation on the development of autoimmune B-cell phenotypes and autoantibodies in the BXD2 murine model of lupus using a cohort of backcrossed progeny (BXD2 x C57BL/6J) x BXD2. Sex was the key factor leading to increased total IgG, IgG2b, and autoantibodies. The percentage of T-bet+CD11c+ IgD+ activated naive B cells (aNAV) was higher in females and was associated with increased T-bet+CD11c+ IgD− age-related B cells, Fas+GL7+ germinal center B cells, Cxcr5−Icos+ peripheral T-helper cells, and Cxcr5+Icos+ follicular T-helper cells. IFN-β was elevated in females. Variation in aNAV cells was mapped to Chr 7 in a locus that shows significant interactions between the female sex and heterozygous B/D variant. Our results suggest that activation of naive B cells forms the basis for the female-predominant development of autoantibodies in lupus-susceptible BXD2 mice.

We recently showed that in systemic lupus erythematosus (SLE), IL-4R signaling is a powerful antagonist that can effectively suppress the development of activated naïve (aNAV) and CD11c +T-bet +IgD −CD27 −double negative 2 (DN2) B cells promoted by both type I and type II IFNs. In the present study, we used the BXD2 mouse model of lupus to determine the mechanism of IL-4 in suppressing the development of DN2 B cells in vivo. Administration of IL-4 significantly inhibited the development of anti-Smith, anti-DNA, and anti-histone autoantibodies induced by TLR7 agonist R848 in BXD2 mice. This was associated with a decreased percentages of CD11c +T-bet +IgD −B cells. Feature-barcoding single cell RNA-sequencing analysis showed that IL-4 modulated B cell development at the transitional stage 2 (T2) and skewed naïve B cells to develop into the CD23 +CD21 −follicular B cells. IL-4 induced the gene encoding interleukin-4-induced1 (IL4i1), an enzyme that metabolizes aromatic amino acids and this was associated with the upregulation of aryl hydrocarbon receptor (AhR) and downstream genes. Metabolomics analysis revealed IL-4 induced AhR agonistic metabolites in B cells including kynurenine (Kyn), indole-3-acetic acid, and indole-3-lactic acid. In the absence of IL-4, Kyn and a potent AhR agonist, formylindolo[3,2-b]carbazole (FICZ), significantly suppressed TLR7 plus IFNβ-induced DN2 B-cell development in vitro. Our results suggest that IL-4 acts through the IL4i1-AhR pathway to inhibit B-cell regulatory response to TLR7 and type I IFN. Identification of small molecular metabolites that act directly in B cells to induce homeostasis may lead to development of orally dosed metabolome modulating therapeutics efficacious in the treatment of SLE. This study was supported by grants from VA Merit Review grant (I01BX004049), NIH grants R01 AI134023, and Lupus Research Alliance Distinguished Innovator Award to J.D.M, the LRA Target Identification in Lupus Award to H-C.H., and the P30-AR-048311.

Background Pathogenic extrafollicular double negative 2 (DN2) B cells in SLE have a phenotype of IgD−CD27−Tbet+CD11c+, and are the precursor of ribonuclear protein (RNP) autoantibody producing B cells. This trajectory of development is promoted by a synergistic effect of type I interferon (IFN) and TLR7-induced activation of B cells at the transitional (Tr) and naïve (NAV) developmental stages. In contrast, IL-4 and IL-4R expression is low in SLE patients and higher expression of IL-4 has been associated with a milder disease course in SLE. The present studies were carried out to determine the mechanism associated with IL-4- mediated B-cell quiescence program in vitro in SLE B cells and in vivo in the BXD2 mouse model of SLE. Methods All SLE patients met the American College of Rheumatology 1997 revised criteria and the 2017 ACR/EULAR classification criteria for SLE. Peripheral blood mononuclear cells were analyzed by FACS for surface expression of IL-4R, intracellular IFN-β, and intranuclear T-bet and IRF7. DN2 B cell differentiation in vitro was stimulated with or without IL-4 50 ng/ml pre-culture for 1 hr. B-cell phenotypes, autoantibody profiles, and their association with the expression of IL-4R and IFN-β were analyzed in 47 SLE patients. The transcriptomics program associated with B-cell fate decision at the Tr, NAV, and activated naïve (aNAV) stages of B cell development in healthy control (HC) subjects and SLE subjects was analyzed using single cell RNA-sequencing (scRNA-seq) analysis. BXD2 mice were injected weekly with IL-4 complex or carrier anti-IL-4 antibody followed by R848, a TLR7 ligand. Four weeks later, mice were treated an additional IV injection of IL-4 complex or carrier. One week later, mice were sacrificed. The development of autoantibodies, GC B cells, DN2 B cells, and B-cell transcriptome were analyzed. Results Section scRNA-seq analysis showed that type I interferon (IFN) stimulated genes (ISGs) were upregulated at Tr, rNAV, and aNAV stages of SLE B cells. In contrast, Tr and NAV B cells from SLE patients exhibited downregulation of an IL-4R quiescent gene program consisting of IL4R, BACH2, and FCRE2A (CD23). In HC, aNAV B cells exhibited upregulation of gene signatures of germinal center (GC) and classical memory (cMEM) B cells including LTB, GPR183, CD27, CD44, and CD83. IKAROS was identified as a top transcription factor associated with the upregulated genes in B cells from HC. In contrast, in SLE, aNAV B cells expressed signature genes of DN2 B cells including FCRL3, FCRL5, and ZEB2. Pseudotime analysis revealed in SLE, 63% of B cells developed into the DN2 pathway compared to only 3% in healthy controls. In contrast, only 14% of SLE B cells developed into the GC and classical memory pathway compared to 20% for healthy controls. IL-4 pre-treatment resulted in a significant increase of the CD11c−Tbet− DN1 subpopulation and a decrease in the CD11c+Tbet+ DN2 B cells from SLE patients. There was a significant correlation of the percent of IL4R−IFNβ+ naïve B cells with SLEDAI, anti-Sm and anti-DNA. In vivo pre-treatment of BXD2 mice with IL-4 significantly blocked R848 induction of CD11c+CD21−Tbet+ DN2 B cells in the spleen. This was associated with a significant decrease in anti-DNA, anti-histone, anti-Sm, and anti-RNP autoantibodies. scRNA-seq analysis revealed that the molecular mechanism for IL-4 suppression of the R848 response was mediated through an transcriptome of aerobic metabolism and an IL-4-induced 1 (IL4i1)-Aryl hydrocarbon receptor (AhR) pathway. IL-4 inhibition of DN2 B cell development in human B cells in vitro was partially inhibited by the AhR inhibitor CH-223191. Conclusion Low expression of the IL-4R program and low signaling through IL-4R at the Tr and naïve B cell stages in SLE pre-disposes such B cells to activation through TLR7. This can upregulate signaling through type II interferon and along with other stimuli to promote development of pathogenic DN2 B cells. Development of DN2 B cells can also be inhibited by IL-4R pathway agonist including treatment of cells with IL4i1 or other molecular activators of the AhR or tryptophan metabolites, such as IAA and IALD. Further studies of these pathways and molecules that can effectively and beneficially modify them could lead to improved treatment for SLE. LAY ABSTRACT SLE is associated with excessive activation of lymphocytes, primarily B lymphocytes that can produce autoantibodies and pathogenic cytokines. Excessive B cell activation in SLE can be linked to several activation molecules including type I interferon, type II interferon, and cytokines including IL-17 and others. Much of the focus for treatment of SLE has been on neutralizing the pathogenic cytokines that drive the development of pathogenic B cells. In contrast, very little attention has been directed towards understanding how B cells in SLE patients become susceptible to activation through diverse pathways, and if lowered levels of immune mediators that maintain B lymphocyte in a quiescence status can contribute to the initiation of the autoimmune process. Previous studies are shown that while most of the inflammatory cytokines and factors are upregulated in SLE, IL-4 and its receptor (IL4R) are consistently downregulated in SLE patients. The present results have studied the effects of the low expression of IL-4R in the initial stages of B cell development in SLE. We have identified that IL-4R exhibited low expression whereas type I interferon exhibited high expression in SLE patients compared to normal controls. This imbalance in IL-4 and type I interferon resulted in a differential gene program regulation of B cells that prompts the activation and development of B cells into a pathogenic "DN2" population of B cells. In contrast, in normal controls, this IL-4R programed naïve B cell development promotes a different B-cell developmental trajectory that do not result in production of autoantibodies. The effects of IL-4 inhibiting pathogenic B cell development was analyzed in humans and in a mouse model of lupus. In both cases, pre- treatment with IL-4 blocked TLR7 and type I interferon-induced pathogenic "DN2" B cell development and suppressed the circulating levels of autoantibodies that are commonly seen in SLE patients. The molecular mechanism for IL-4R suppression of development of pathogenic B cells in lupus was found to be based on key molecules that regulate metabolism and quiescence of B cells. Further studies of these pathways and molecules that can effectively and beneficially modify them could lead to improved treatment for SLE.

Abnormal expansion of type I interferon (IFN) stimulated B cells and T-bet+CD11c+IgD−CD27− DN2 atypical memory B cells are both hallmarks of SLE. To determine how they are interrelated, we have carried out single cell transcriptomics analysis using B cells derived from 3 SLE patients (anti-DNA+ and anti-Smith+) and 3 healthy controls (HCs)(all African Americans). We have identified that elevation of IL4R in pre-switched IGHM+/IGHD+ B cells is the opposing signature of type I IFN stimulated genes (ISGs) and DN2 B cells (TBX21, ITGAX, FCRL3, FCRL5, and ZEB2). In HCs, IGHM+/IGHD+ B cells expressed elevated IL4R. Further, activated naïve B cells (IGHM+IGHD+IGHG+) exhibited upregulation of the germinal center-oriented classical memory B-cell signature genes, CD27, TNFRSF13B (the gene encoding TACI), and GPR183 (the gene encoding EBI2). Flow cytometry analysis confirmed that down-regulation IL-4R and upregulation of intracellular IFN-β in naïve IgD+CD27− B cells correlated with the accumulation of atypical CD21−IgD−CD27−B cells and a reduction in the percentage of classical IgD−CD27+memoryB cells in SLE patients (n=47). In vitro exposure of SLE B cells to IL-4 prior to treatment with anti-Ig+IL-21+IFN-gamma+TLR7+BAFF stimulation significantly enhanced B-cell development into CD27+CD38+ plasmablasts/plasma cells as well as IgD−CD27+memoryB cells but it significantly blocked the development of DN2 B cells. Our results show that upregulation of type I IFN and down-regulation of IL-4R signaling in naïve B cells lead to the accumulation of atypical memory B cells in SLE. Type I IFN and IL-4R signaling in naïve B cells induce the development of distinct lineages of atypical versus classical memory B cells, respectively.

IL-17 receptor A (IL-17RA) expression has been identified in B cells and macrophages. There was low expression of IL-17RA on T cells. Surprisingly, we previously showed that IL-17RA signaling through NF-κB p65/p50 has a complex effect on both B and T-cell localization and germinal center (GC) architecture, making it difficult to dissect the specific effects of IL-17RA on B cells. To isolate the effects of the absence of IL-17RA specifically on B cells, we have generated a B-cell specific IL-17RA knockout (Il17rafloxed/floxed x Cd19.Cre) BXD2 mice. Flow cytometry confirmed the loss of IL-17RA expression in only CD19+ B cells but not CD3+ T cells or CD11b+ macrophages. There was a 2-fold decreased spontaneous GC B cell population in Il17rafloxed/floxed x Cd19.Cre BXD2, compared to Il17rawt/floxed x Cd19.Cre BXD2 mice. Interestingly, the abnormal activation and expansion of T-follicular helper cell (Tfh) phenotype in BXD2-Il17ra−/− mice was abrogated in Il17rafloxed/floxed x Cd19.Cre BXD2 mice. In the BXD2-Il17ra−/− mice, B cells were anergic to TLR7 stimulation and exhibited a strong stimulus-specific transcription repressor p50/p50 homodimer and a lower NF-κB phospho-p65. To demonstrate that this anergy is due specifically to IL-17RA on B cells but not other cells, we have identified that there was a lower TLR7-induced CD69 in vitro in Il17rafloxed/floxed x Cd19.Cre BXD2 mice. Taken together, our results suggest that IL-17RA signaling directly regulates B cell autoimmune phenotype in the BXD2 mice through an enhancement of B-cell responses stimulated by the NF-κB signaling pathway. A deficiency of such signal enforces B cell anergy and prevents autoreactive GC formation.

Systemic Lupus Erythematosus (SLE) is characterized by high level of type I interferon (IFN) and abnormal expansion of effector B cells including IgD−CD27−double negative (DN), CD21−T-bet+DN2 B cells, and antibody secreting cells (ASCs). It remains unclear as to the role of type I and type II interferon (IFN) to promote DN2 B cell differentiation. The in vivo effects of type I IFN signaling in promoting DN B cells were accessed using the BXD2 mouse model of lupus which exhibit a low Th1 response, compared to normal B6 mice. Compared to normal B6 mouse, there was a significantly elevated percent of DN B cells in the BXD2 mice, but this was reduced in BXD2 mice that are deficient in IFNAR1 (BXD2-Ifnar1−/−). Compared to wild-type BXD2 mice, there was a lower level of IgG autoantibodies specific to DNA, histone and Smith (Sm) in BXD2-Ifnar1−/− mice. BXD2-Ifnar1−/− B cells presented impaired differentiation into the DN and the DN2 populations under an in vitro stimulation condition in the presence of IFNb and a TLR7 agonist. To determine if type I IFN stimulation can direct differentiation of healthy B cells to these effector B cells without the influence of type II IFN, B cells purified from healthy individuals were stimulated under a DN stimulation condition (anti-Ig+IL-21+BAFF+TLR7) in the presence and absence of IFNb. We found that IFNb significantly increased the percentage and of DN and DN2 B cells, but decreased the percentage of CD27+CD38+ plasmablasts/plasma. Under the same condition, IFNb increased the secretion of autoantibodies specific to DNA, histone and Sm. Our results suggest that type I IFN signaling stimulates DN2 B cell differentiation and can promote the abnormal expansion of these B cells in vivo independent of type II IFN.

Abstract Programmed cell death 1 ligand 1 (PD-L1) is a key driver of tumor-mediated immune suppression, and targeting it with antibodies can induce therapeutic responses. Given the costs and associated toxicity of PD-L1 blockade, alternative therapeutic strategies are needed. Using reverse-phase protein arrays to assess drugs in use or likely to enter trials, we performed a candidate drug screen for inhibitors of PD-L1 expression and identified verteporfin as a possible small-molecule inhibitor. Verteporfin suppressed basal and IFN-induced PD-L1 expression in vitro and in vivo through Golgi-related autophagy and disruption of the STAT1–IRF1–TRIM28 signaling cascade, but did not affect the proinflammatory CIITA-MHC II cascade. Within the tumor microenvironment, verteporfin inhibited PD-L1 expression, which associated with enhanced T-lymphocyte infiltration. Inhibition of chromatin-associated enzyme PARP1 induced PD-L1 expression in high endothelial venules (HEV) in tumors and, when combined with verteporfin, enhanced therapeutic efficacy. Thus, verteporfin effectively targets PD-L1 through transcriptional and posttranslational mechanisms, representing an alternative therapeutic strategy for targeting PD-L1.

OBJECTIVES:Enamel organ epithelium (EOE) gives rise to the major epithelial-derived cell types of tooth including the ameloblasts. The formation of enamel, termed amelogenesis, occurs through the cytodifferentiation of ameloblasts, ultimately leading to apoptosis and necrosis of these cells with eruption. Therefore, studies regarding enamel matrix formation and bioengineering have been limited. In this study, we establish and characterize two mouse immortalized ameloblast-like cell lines using human papillomavirus 16 (HPV16) E6/E7 oncogenes for the first time.SETTING AND SAMPLE POPULATION:Two mouse EOE dental cell lines (EOE-2M and EOE-3M).MATERIAL AND METHODS:Isolated EOE primary cells were used to establish clonal cell lines and immortalized using the HPV16 E6/E7 gene platform. Two established cell lines were characterized by growth rate (Cell Proliferation Assay, MTS), gene (quantitative RT-PCR) and protein (immunocytochemistry) expression profiles, and mineralization potential (in situ alkaline phosphatase (ALP) histochemistry and Xylene Orange staining) in media supplemented with ascorbic acid and β-glycerophosphate. Gene and protein expression analyses included specific enamel matrix and ameloblast cell markers: Amel, Ambn, Enam, Amtn, ODAM, MMP20, Krt14 and DLX3.RESULTS:Both cell lines were maintained in excess of 30 passages, with EOE-3M cells proliferating at a slightly higher rate. The cell lines expressed all tested enamel matrix markers and produced a mineralized ECM demonstrating an ameloblast-like profile.CONCLUSIONS:Two mouse ameloblasts-like immortalized cell lines have been characterized that will be useful tool for studies regarding enamel bioengineering.

Cleidocranial dysplasia (CCD) is an autosomal dominant disorder affecting osteoblast differentiation, chondrocyte maturation, skeletal morphogenesis, and tooth formation. Dental phenotype in CCD include over-retained primary teeth, failed eruption of permanent teeth, and supernumerary teeth. The underlying mechanism is unclear. We previously reported one CCD patient with allelic RUNX2 deletion (CCD-011). In the current study, we determined the transcriptomic profiles of dental pulp cells from this patient compared to one sex-and-age matched non-affected individual. Next Generation RNA sequencing revealed that 60 genes were significantly dysregulated (63% upregulated and 27% downregulated). Among them, IGFBP2 (insulin-like growth factor binding protein-2) was found to be upregulated more than twofold in comparison to control cells. Stable overexpression of RUNX2 in CCD-011 pulp cells resulted in the reduction of IGFBP2. Moreover, ALPL expression was up-regulated in CCD-011 pulp cells after introduction of normal RUNX2. Promoter analysis revealed that there are four proximal putative RUNX2 binding sites in -1.5 kb IGFBP2 promoter region. Relative luciferase assay confirmed that IGFBP2 is a direct target of RUNX2. Immunohistochemistry demonstrated that IGFBP2 was expressed in odontoblasts but not ameloblasts. This report demonstrated the importance of RUNX2 in the regulation of gene profile related to dental pulp cells and provided novel insight of RUNX2 into the negative regulation of IGFBP2.

Singleton-Merten syndrome (SMS) is an autosomal dominant, multi-system innate immune disorder characterized by early and severe aortic and valvular calcification, dental and skeletal abnormalities, psoriasis, glaucoma, and other varying clinical findings. Recently we identified a specific gain-of-function mutation in IFIH1, interferon induced with helicase C domain 1, segregated with this disease. SMS disease without hallmark dental anomalies, termed atypical SMS, has recently been reported caused by variants in DDX58, DEXD/H-box helicase 58. IFIH1 and DDX58 encode retinoic acid-inducible gene I (RIG-I)-like receptors family members melanoma differentiation-associated gene 5 and RIG-I, respectively. These cytosolic pattern recognition receptors function in viral RNA detection initiating an innate immune response through independent pathways that promote type I and type III interferon expression and proinflammatory cytokines. In this review, we focus on SMS as an innate immune disorder summarizing clinical features, molecular aspects of the pathogenetic pathway and discussing underlying mechanisms of the disease.

Abstract Purpose: In prostate cancer cells, there is CD24-dependent inactivation of mutant p53, but the mechanism and its significance remain largely unknown. Here, we validated this observation and explored the therapeutic potential of targeting CD24 in TP53 mutant prostate cancer cells. Experimental Design: Overall, 553 prostate cancers (522 formalin-fixed paraffin-embedded and 31 frozen tissues) were assessed for protein or mRNA expression of CD24 and TP53. The effects of CD24 on p53-dependent transcriptional regulation, cancer cell growth, the cell cycle, apoptosis, and mutant p53 restoration were also determined. Results: As determined with three sample cohorts, CD24 and p53 were not expressed in prostate epithelial cells but in prostate cancer cells in 48% of cases for CD24 and 16% of cases for p53 (mutant form). Expressions of CD24 and mutant p53 were more frequently observed in late-stage and metastatic prostate tumors. Mutant p53 accompanied with CD24 was expressed in most cases (91.6%, 76/83). Silencing of CD24 increased the transcriptional activity of p53 target genes, such as CDKNA1, VDR, and TP53INP1, leading to suppression of p53-dependent cell growth, cell-cycle arrest, and apoptosis in most TP53-mutant prostate cancer cells. Silencing of CD24 enhanced restoration of PRIMA-1–induced mutant p53 in endogenous TP53P223L/V274F DU145 cells and in PC3 cells transfected with TP53R273H. Conclusions: In human prostate cancers, there is CD24-dependent inactivation of mutant p53. The coexpression of CD24 and p53 may help identify aggressive cancers. Targeting CD24 provides a strategy to enhance mutant p53-restoring therapies, especially in patients with TP53R273H prostate cancer. Clin Cancer Res; 22(10); 2545–54. ©2015 AACR.

The Apert syndrome is a rare congenital disorder most often arising from S252W or P253R mutations in fibroblast growth factor receptor (FGFR2). Numerous studies have focused on the regulatory role of Apert FGFR2 signaling in bone formation, whereas its functional role in tooth development is largely unknown. To investigate the role of FGFR signaling in cell proliferation and odontogenic differentiation of human dental cells in vitro, we isolated dental pulp and enamel organ epithelia (EOE) tissues from an Apert patient carrying the S252W FGFR2 mutation. Apert primary pulp and EOE cells were established and shown to exhibit normal morphology and express alkaline phosphatase under differentiation conditions. Similar to control cells, Apert dental pulp and EOE cells expressed all FGFRs, with highest levels of FGFR1 followed by FGFR2 and low levels of FGFR3 and FGFR4. However, Apert cells had increased cell growth compared with control cells. Distinct from previous findings in osteoblast cells, gain-of-function S252W FGFR2 mutation did not upregulate the expression of epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFRα), but elevated extracellular signal-regulated kinase (ERK) signaling in cells after EGF stimulation. Unexpectedly, there was little effect of the S252W mutation on odontogenic gene expression in dental pulp and EOE cells. However, after inhibition of total FGFR signaling or ERK signaling, the expression of odontogenic genes was upregulated in both dental cell types, indicating the negative effect of whole FGFR signaling on odontogenic differentiation. This study provides novel insights on FGFR signaling and a common Apert FGFR2 mutation in the regulation of odontogenic differentiation of dental mesenchymal and epithelial cells.