Systemic lupus erythematosus is considered to be under the control of polygenic inheritance, developing according to the cumulative effects of susceptibility genes with polymorphic alleles; however, the mechanisms underlying the roles of polygenes based on functional and pathological genomics remain uncharacterized. In this study, we substantiate that a CD72 polymorphism in the membrane-distal extracellular domain impacts on both the development of glomerulonephritis and vasculitis in a lupus model strain of mice, MRL/MpJ-Faslpr, and the reactivity of BCR signal stimulation. We generated mice carrying a bacterial artificial chromosome transgene originating from C57BL/6 (B6) mice that contains the Cd72b locus (Cd72B6 transgenic [tg]) or the modified Cd72b locus with an MRL-derived Cd72c allele at the polymorphic region corresponding to the membrane-distal extracellular domain (Cd72B6/MRL tg). Cd72B6 tg mice, but not Cd72B6/MRL tg mice, showed a significant reduction in mortality following a marked improvement of disease associated with decreased serum levels of IgG3 and anti-dsDNA Abs. The number of splenic CD4−CD8− T cells in Cd72B6 tg mice was decreased significantly in association with a reduced response to B cell receptor signaling. These results indicate that the Cd72 polymorphism affects susceptibility to lupus phenotypes and that novel functional rescue by a bacterial artificial chromosome transgenesis is an efficient approach with wide applications for conducting a genomic analysis of polygene diseases.

In humans, genetic variation and dietary factors may alter the biologic effects of exposure to 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), one of the major heterocyclic amines generated from cooking meats at high temperatures that has carcinogenic potential through the formation of DNA adducts. Previously, we reported grilled red meat consumption associated with PhIP-DNA adduct levels in human prostate. In the present study, we expanded our investigation to estimate the associations between beverage consumption and PhIP-DNA adduct levels in prostate for 391 prostate cancer cases. Of the 15 beverages analyzed, red wine consumption had the strongest association with PhIP-DNA adduct levels showing an inverse correlation in both tumor (p=0.006) and non-tumor (p=0.002) prostate cells. Red wine consumption differed significantly between African-American and white cases, but PhIP-DNA adduct levels in prostate did not vary by race. In African Americans compared with whites, however, associations between red wine consumption and PhIP-DNA adduct levels were not as strong as associations with specific (e.g., SULT1A1 and UGT1A10 genotypes) and non-specific (e.g., African ancestry) genetic variation. In a multivariable model, the covariate for red wine consumption explained a comparable percentage (13-16%) of the variation in PhIP-DNA adduct levels in prostate across the two racial groups, but the aforementioned genetic factors explained 33% of the PhIP-DNA adduct variation in African-American cases, while only 19% of the PhIPDNA adduct variation in whites. We conclude that red wine consumption may counteract biologic effects of PhIP exposure in human prostate, but genetic factors may play an even larger role, particularly in African Americans.

Cyclin-dependent kinase 5 (Cdk5) plays a pivotal role in neuronal migration and differentiation, and in axonal elongation. Although many studies have been conducted to analyze neuronal functions of Cdk5, its kinase activity has also been reported during oligodendrocyte differentiation, which suggests Cdk5 may play an important role in oligodendrocytes. Here, we describe a hypomyelination phenotype observed in Emx1-cre mediated Cdk5 conditional knockout (cKO) mice (Emx1-cKO), in which the Cdk5 gene was deleted in neurons, astrocytes and oligodendrocyte -lineage cells. In contrast, the Cdk5 gene in CaMKII cKO mice was deleted only in neurons. Because the development of mature oligodendrocytes from oligodendrocyte precursor cells is a complex process, we performed in situ hybridization using markers for the oligodendrocyte precursor cell and for the differentiated oligodendrocyte. Our results indicate that hypomyelination in Emx1-cKO is due to the impaired differentiation of oligodendrocytes, rather than to the proliferation or migration of their precursors. The present study confirmed the in vivo role of Cdk5 in oligodendrocyte differentiation.

During vertebrate lens development, the anterior, ectoderm-derived lens vesicle cells differentiate into a monolayer of epithelial cells that retain proliferative potential. Subsequently, they exit the cell cycle and give rise to posterior lens fiber cells that form the lens body. In the present study, we demonstrate that the transcription factor GATA-3 is expressed in the posterior lens fiber cells during embryogenesis, and that GATA-3-deficiency impairs lens development. Interestingly, expression of E-cadherin, a premature lens vesicle marker, is abnormally prolonged in the posterior region of Gata3 homozygous mutant lenses. Furthermore, expression of γ-crystallin, a differentiation marker for fiber cells, is reduced. This suppressed differentiation is accompanied by an abnormal cellular proliferation, as well as with diminished levels of the cell-cycle inhibitors Cdkn1b/p27 and Cdkn1c/p57 and increased Ccnd2/cyclin D2 abundance. Thus, these observations suggest that GATA-3 is essential for lens cells differentiation and proper cell cycle control.

During the differentiation of the mammalian embryonic testis, two compartments are defined: the testis cords and the interstitium. The testis cords give rise to the adult seminiferous tubules, whereas steroidogenic Leydig cells and other less well characterized cell types differentiate in the interstitium (the space between testis cords). Although the process of testis cord formation is essential for male development, it is not entirely understood. It has been viewed as a Sertoli-cell driven process, but growing evidence suggests that interstitial cells play an essential role during testis formation. However, little is known about the origin of the interstitium or the molecular and cellular diversity within this early stromal compartment. To better understand the process of mammalian gonad differentiation, we have undertaken an analysis of developing interstitial/stromal cells in the early mouse testis and ovary. We have discovered molecular heterogeneity in the interstitium and have characterized new markers of distinct cell types in the gonad: MAFB, C-MAF, and VCAM1. Our results show that at least two distinct progenitor lineages give rise to the interstitial/stromal compartment of the gonad: the coelomic epithelium and specialized cells along the gonad-mesonephros border. We demonstrate that both these populations give rise to interstitial precursors that can differentiate into fetal Leydig cells. Our analysis also reveals that perivascular cells migrate into the gonad from the mesonephric border along with endothelial cells and that these vessel-associated cells likely represent an interstitial precursor lineage. This study highlights the cellular diversity of the interstitial cell population and suggests that complex cell-cell interactions among cells in the interstitium are involved in testis morphogenesis.

Background

Deficiency of the transcription factor MafB, which is normally expressed in macrophages, can underlie cellular dysfunction associated with a range of autoimmune diseases and arteriosclerosis. MafB has important roles in cell differentiation and regulation of target gene expression; however, the mechanisms of this regulation and the identities of other transcription factors with which MafB interacts remain uncertain. Bioinformatics methods provide a valuable approach for elucidating the nature of these interactions with transcriptional regulatory elements from a large number of DNA sequences. In particular, identification of patterns of co-occurrence of regulatory cis-elements (motifs) offers a robust approach.

Results

Here, the directional relationships among several functional motifs were evaluated using the Log-linear Graphical Model (LGM) after extraction and search for evolutionarily conserved motifs. This analysis highlighted GATA-1 motifs and 5’AT-rich half Maf recognition elements (MAREs) in promoter regions of 18 genes that were down-regulated in Mafb deficient macrophages. GATA-1 motifs and MafB motifs could regulate expression of these genes in both a negative and positive manner, respectively. The validity of this conclusion was tested with data from a luciferase assay that used a C1qa promoter construct carrying both the GATA-1 motifs and MAREs. GATA-1 was found to inhibit the activity of the C1qa promoter with the GATA-1 motifs and MafB motifs.

Conclusions

These observations suggest that both the GATA-1 motifs and MafB motifs are important for lineage specific expression of C1qa. In addition, these findings show that analysis of combinations of evolutionarily conserved motifs can be successfully used to identify patterns of gene regulation.

Newly identified tissue-resident vascular precursor cells are recruited into growing vessels and contribute to vasculogenesis in adult mice.

Vasculogenesis describes the process of de novo vessel formation from vascular precursor cells. Although formation of the first major vessels, such as the dorsal aorta and cardinal veins, occurs during embryonic vasculogenesis, the contribution of precursor cell populations to postnatal vessel development is not well understood. Here, we identified a novel population of postnatal vascular precursor cells in mice. These cells express the Schwann cell protein myelin protein zero (Po) and exhibit a CD45−CD31−VEcad−c-kit+CXCR4+ surface phenotype. Po+ vascular precursors (PVPs) are recruited into the growing vasculature, and comprise a minor population of arterial endothelial cells in adult mice. Recruitment of PVPs into growing vessels is mediated by CXCL12–CXCR4 signaling, and is enhanced during vascular expansion induced by Notch inhibition. Po-specific ablation of Flk1, a receptor for VEGF, results in branching defects and insufficient arterial patterning in the retina, as well as reduced neovascularization of tumors and ischemic tissues. Thus, in postnatal mice, although growing vessels are formed primarily by angiogenesis from preexisting vessels, a minor population of arterial endothelia may be derived from tissue-resident vascular precursor cells.

Protein ubiquitination is a post-translational protein modification that regulates many biological conditions [1], [2], [3], [4]. Trip12 is a HECT-type E3 ubiquitin ligase that ubiquitinates ARF and APP-BP1 [5], [6]. However, the significance of Trip12 in vivo is largely unknown. Here we show that the ubiquitin ligase activity of Trip12 is indispensable for mouse embryogenesis. A homozygous mutation in Trip12 (Trip12mt/mt) that disrupts the ubiquitin ligase activity resulted in embryonic lethality in the middle stage of development. Trip12mt/mt embryos exhibited growth arrest and increased expression of the negative cell cycle regulator p16 [7], [8], [9], [10]. In contrast, Trip12mt/mt ES cells were viable. They had decreased proliferation, but maintained both the undifferentiated state and the ability to differentiate. Trip12mt/mt ES cells had increased levels of the BAF57 protein (a component of the SWI/SNF chromatin remodeling complex) and altered gene expression patterns. These data suggest that Trip12 is involved in global gene expression and plays an important role in mouse development.

OBJECTIVE

Tissue-specific self-antigens are ectopically expressed within the thymus and play an important role in the induction of central tolerance. Insulin is expressed in both pancreatic islets and the thymus and is considered to be the primary antigen for type 1 diabetes. Here, we report the role of the insulin transactivator MafA in the expression of insulin in the thymus and susceptibility to type 1 diabetes.

RESEARCH DESIGN AND METHODS

The expression profiles of transcriptional factors (Pdx1, NeuroD, Mafa, and Aire) in pancreatic islets and the thymus were examined in nonobese diabetic (NOD) and control mice. Thymic Ins2 expression and serum autoantibodies were examined in Mafa knockout mice. Luciferase reporter assay was performed for newly identified polymorphisms of mouse Mafa and human MAFA. A case-control study was applied for human MAFA polymorphisms.

RESULTS

Mafa, Ins2, and Aire expression was detected in the thymus. Mafa expression was lower in NOD thymus than in the control and was correlated with Ins2 expression. Targeted disruption of MafA reduced thymic Ins2 expression and induced autoantibodies against pancreatic islets. Functional polymorphisms of MafA were newly identified in NOD mice and humans, and polymorphisms of human MAFA were associated with susceptibility to type 1 diabetes but not to autoimmune thyroid disease.

CONCLUSIONS

These data indicate that functional polymorphisms of MafA are associated with reduced expression of insulin in the thymus and susceptibility to type 1 diabetes in the NOD mouse as well as human type 1 diabetes.

Cdk5 plays a role in nervous system development; its role in the initial stages of neural differentiation is poorly understood. We isolated neural stem cells from E13 Cdk5 WT and KO mouse and observed them as they switched from proliferating stage to neural differentiation. We show that Cdk5 phosphorylation of p27kip1 at Thr187 is crucial to neural differentiation.

Cyclin-dependent kinase 5 (Cdk5) plays a key role in the development of the mammalian nervous system; it phosphorylates a number of targeted proteins involved in neuronal migration during development to synaptic activity in the mature nervous system. Its role in the initial stages of neuronal commitment and differentiation of neural stem cells (NSCs), however, is poorly understood. In this study, we show that Cdk5 phosphorylation of p27Kip1 at Thr187 is crucial to neural differentiation because 1) neurogenesis is specifically suppressed by transfection of p27Kip1 siRNA into Cdk5+/+ NSCs; 2) reduced neuronal differentiation in Cdk5−/− compared with Cdk5+/+ NSCs; 3) Cdk5+/+ NSCs, whose differentiation is inhibited by a nonphosphorylatable mutant, p27/Thr187A, are rescued by cotransfection of a phosphorylation-mimicking mutant, p27/Thr187D; and 4) transfection of mutant p27Kip1 (p27/187A) into Cdk5+/+ NSCs inhibits differentiation. These data suggest that Cdk5 regulates the neural differentiation of NSCs by phosphorylation of p27Kip1 at theThr187 site. Additional experiments exploring the role of Ser10 phosphorylation by Cdk5 suggest that together with Thr187 phosphorylation, Ser10 phosphorylation by Cdk5 promotes neurite outgrowth as neurons differentiate. Cdk5 phosphorylation of p27Kip1, a modular molecule, may regulate the progress of neuronal differentiation from cell cycle arrest through differentiation, neurite outgrowth, and migration.

Candida species are the most common source of nosocomial invasive fungal infections. Previous studies have indicated that T-helper immune response is the critical host factor for susceptibility to Candida infection. The transcription factor GATA-3 is known as the master regulator for T-helper type 2 (Th2) differentiation. We therefore investigated the role of GATA-3 in the host defense against systemic Candida infection using GATA-3-overexpressing transgenic mice. The survival of GATA-3-overexpressing mice after Candida infection was significantly lower than that of wild-type mice. Candida outgrowth was significantly increased in the kidneys of GATA-3-overexpressing mice, compared with wild-type mice. The levels of various Th2 cytokines, including interleukin-4 (IL-4), IL-5, and IL-13, were significantly higher while the level of Th1 cytokine gamma interferon was significantly lower in the splenocytes of GATA-3-overexpressing mice after Candida infection. Recruitment of macrophages into the peritoneal cavity in response to Candida infection and their phagocytic activity were significantly lower in GATA-3-overexpressing mice than in wild-type mice. Exogenous administration of gamma interferon to GATA-3-overexpressing mice significantly reduced Candida outgrowth in the kidney and thus increased the survival rate. Administration of gamma interferon also increased the recruitment of macrophages into the peritoneal cavity in response to Candida infection. These results indicate that overexpression of GATA-3 modulates macrophage antifungal activity and thus enhances the susceptibility to systemic Candida infection, possibly by reducing the production of gamma interferon in response to Candida infection.

Aging leads to the disruption of the homeostatic balance of multiple biological systems. In bone marrow multipotent mesenchymal cells undergo differentiation into various anchorage-dependent cell types, including osteoblasts and adipocytes. With age as well as with treatment of antidiabetic drugs such as thiazolidinediones, mesenchymal cells favor differentiation into adipocytes, resulting in an increased number of adipocytes and a decreased number of osteoblasts, causing osteoporosis. The mechanism behind this differentiation switch is unknown. Here we show an age-related decrease in the expression of Maf in mouse mesenchymal cells, which regulated mesenchymal cell bifurcation into osteoblasts and adipocytes by cooperating with the osteogenic transcription factor Runx2 and inhibiting the expression of the adipogenic transcription factor Pparg. The crucial role of Maf in both osteogenesis and adipogenesis was underscored by in vivo observations of delayed bone formation in perinatal Maf–/– mice and an accelerated formation of fatty marrow associated with bone loss in aged Maf+/– mice. This study identifies a transcriptional mechanism for an age-related switch in cell fate determination and may provide a molecular basis for novel therapeutic strategies against age-related bone diseases.

The glomerular basement membrane (GBM) is a key component of the filtering unit in the kidney. Mutations involving any of the collagen IV genes (COL4A3, COL4A4, and COL4A5) affect GBM assembly and cause Alport syndrome, a progressive hereditary kidney disease with no definitive therapy. Previously, we have demonstrated that the bone morphogenetic protein (BMP) antagonist uterine sensitization–associated gene-1 (USAG-1) negatively regulates the renoprotective action of BMP-7 in a mouse model of tubular injury during acute renal failure. Here, we investigated the role of USAG-1 in renal function in Col4a3–/– mice, which model Alport syndrome. Ablation of Usag1 in Col4a3–/– mice led to substantial attenuation of disease progression, normalization of GBM ultrastructure, preservation of renal function, and extension of life span. Immunohistochemical analysis revealed that USAG-1 and BMP-7 colocalized in the macula densa in the distal tubules, lying in direct contact with glomerular mesangial cells. Furthermore, in cultured mesangial cells, BMP-7 attenuated and USAG-1 enhanced the expression of MMP-12, a protease that may contribute to GBM degradation. These data suggest that the pathogenetic role of USAG-1 in Col4a3–/– mice might involve crosstalk between kidney tubules and the glomerulus and that inhibition of USAG-1 may be a promising therapeutic approach for the treatment of Alport syndrome.

The present study was undertaken to investigate the effect of dietary supplementation with nimesulide or eugenol on N-nitrosodiethylamine (DEN)-initiated early hepatocarcinogenesis in F344 male rats. Both compounds did not alter the expression of cytochrome P450 (CYP) 2E1, the enzyme that plays a major role in the activation of DEN to genotoxic products; however, nimesulide induced the expression of CYP1A1. Western blot analysis revealed that COX-1 and COX-2 protein expressions were not modulated by DEN compared with normal controls. Furthermore, post-initiation feeding with nimesulide or eugenol did not modulate COX-2 protein expression in normal or DEN-treated rats, whereas eugenol significantly increased the liver prostaglandin E2 (PGE2) levels of DEN-injected animals compared with the DEN controls. Ultimately, nimesulide or eugenol did not modify DEN-induced hepatocarcinogenesis as evidenced by insignificant changes in the number and size of preneoplastic placental glutathione S-transferase (GST-P) positive liver foci compared with the DEN controls. These results suggest that COX-2, as well as prostaglandin E2, may play no role in the post-initiation development of DEN-induced rat hepatocarcinogenesis at an early stage.

The roles of autoimmune regulator (Aire) in the expression of the diverse arrays of tissue-restricted antigen (TRA) genes from thymic epithelial cells in the medulla (medullary thymic epithelial cells [mTECs]) and in organization of the thymic microenvironment are enigmatic. We approached this issue by creating a mouse strain in which the coding sequence of green fluorescent protein (GFP) was inserted into the Aire locus in a manner allowing concomitant disruption of functional Aire protein expression. We found that Aire+ (i.e., GFP+) mTECs were the major cell types responsible for the expression of Aire-dependent TRA genes such as insulin 2 and salivary protein 1, whereas Aire-independent TRA genes such as C-reactive protein and glutamate decarboxylase 67 were expressed from both Aire+ and Aire− mTECs. Remarkably, absence of Aire from mTECs caused morphological changes together with altered distribution of mTECs committed to Aire expression. Furthermore, we found that the numbers of mTECs that express involucrin, a marker for terminal epidermal differentiation, were reduced in Aire-deficient mouse thymus, which was associated with nearly an absence of Hassall's corpuscle-like structures in the medulla. Our results suggest that Aire controls the differentiation program of mTECs, thereby organizing the global mTEC integrity that enables TRA expression from terminally differentiated mTECs in the thymic microenvironment.

Background

Triple-negative breast cancer (estrogen receptor-, progesterone receptor-, and HER2-negative) (TNBC) is a high risk breast cancer that lacks specific therapy targeting these proteins.

Methods

We studied 969 consecutive Japanese patients diagnosed with invasive breast cancer from January 1981 to December 2003, and selected TNBCs based on the immunohistochemical data. Analyses of epidermal growth factor receptor (EGFR) gene mutations and amplification, and BRCA1 mRNA expression were performed on these samples using TaqMan PCR assays. The prognostic significance of TNBCs was also explored. Median follow-up was 8.3 years.

Results

A total of 110 (11.3%) patients had TNBCs in our series. Genotyping of the EGFR gene was performed to detect 14 known EGFR mutations, but none was identified. However, EGFR gene copy number was increased in 21% of TNBCs, while only 2% of ER- and PgR-positive, HER2-negative tumors showed slightly increased EGFR gene copy numbers. Thirty-one percent of TNBCs stained positive for EGFR protein by immunohistochemistry. BRCA1 mRNA expression was also decreased in TNBCs compared with controls. Triple negativity was significantly associated with grade 3 tumors, TP53 protein accumulation, and high Ki67 expression. TNBC patients had shorter disease-free survival than non-TNBC in node-negative breast cancers.

Conclusion

TNBCs have an aggressive clinical course, and EGFR and BRCA1 might be candidate therapeutic targets in this disease.

Recent epidemiologic investigations have observed an association between consumption of grilled or barbequed meat and an increased risk for pancreatic cancer, suggesting that dietary exposure to heterocyclic aromatic amines (HCA) may contribute to the development of this disease. 2-Amino-1-methyl-6-phenylimidazo [4,5-b]-pyridine (PhIP) is the most abundant HCA found in well-done and grilled meats. To determine whether HCA-induced DNA damage is present in the human pancreas, we used immunohistochemistry and computer-assisted image analysis to measure PhIP-DNA adducts in 54 normal pancreatic tissues (N) from persons without pancreatic cancer and in 38 normal adjacent pancreatic tissues (A), and 39 cancer tissues (T) from 68 patients with pancreatic adenocarcinoma. PhIP-DNA adducts were detected in 53 N, 34 A, and 39 T samples. Mean values (±SD) of the absorbency for PhIP staining were 0.22 ± 0.04, 0.24 ± 0.04, and 0.24 ± 0.03 for N, A, and T samples, respectively (p = 0.004). Using the median absorbency (0.21) of the samples from normal controls as the cut-off, 71% of A and 77% of T tissues, compared with 48% of N tissues, were distributed in the higher range (p = 0.009). The odds ratio of pancreatic cancer was 3.4 (95% confidence interval 1.5 to 7.5, p = 0.002) for individuals with a higher level of PhIP-DNA adducts. This is the first report of detection of PhIP-DNA adducts in human pancreatic tissue samples obtained from patients having unknown exposure to HCA. Although limited by the small sample size, these preliminary results suggest that PhIP exposure may contribute to human pancreatic cancer development.

Introduction

Single-nucleotide polymorphisms (SNPs) in codon 72 of the TP53 (also known as p53) gene (rs1042522) and in the promoter region of the MDM2 gene (SNP309; rs2279744) have been suggested to play roles in many cancers. We investigated whether these SNPs were associated with patient outcome and the effect of adjuvant systemic therapy.

Methods

The genotypes of TP53 codon 72 and MDM2 SNP309 were defined among 557 primary Japanese breast cancer patients (median follow-up, 61.7 months). The effects of several variables on survival were tested by Cox's proportional hazards regression analysis.

Results

We showed that the Pro/Pro genotype of TP53 codon 72 was associated with poorer disease-free survival (DFS) than other genotypes by Kaplan-Meier analysis (P = 0.049) and multivariate Cox's proportional hazards regression analysis (P = 0.047, risk ratio of recurrence = 1.67), whereas MDM2 SNP309 status was not associated with DFS. The association of the Pro/Pro TP53 genotype with poorer DFS was especially significant in patients who received adjuvant chemotherapy (P = 0.009). In contrast, among the patients who had received adjuvant hormonal therapy or no adjuvant systemic therapy, TP53 codon 72 genotype was not associated with DFS.

Conclusion

The Pro/Pro genotype of TP53 codon 72 appears to be an independent prognostic marker in breast cancer patients.

MafB is a member of the large Maf family of transcription factors that share similar basic region/leucine zipper DNA binding motifs and N-terminal activation domains. Although it is well known that MafB is specifically expressed in glomerular epithelial cells (podocytes) and macrophages, characterization of the null mutant phenotype in these tissues has not been previously reported. To investigate suspected MafB functions in the kidney and in macrophages, we generated mafB/green fluorescent protein (GFP) knock-in null mutant mice. mafB homozygous mutants displayed renal dysgenesis with abnormal podocyte differentiation as well as tubular apoptosis. Interestingly, these kidney phenotypes were associated with diminished expression of several kidney disease-related genes. In hematopoietic cells, GFP fluorescence was observed in both Mac-1- and F4/80-expressing macrophages in the fetal liver. Interestingly, F4/80 expression in macrophages was suppressed in the homozygous mutant, although development of the Mac-1-positive macrophage population was unaffected. In primary cultures of fetal liver hematopoietic cells, MafB deficiency was found to dramatically suppress F4/80 expression in nonadherent macrophages, whereas the Mac-1-positive macrophage population developed normally. These results demonstrate that MafB is essential for podocyte differentiation, renal tubule survival, and F4/80 maturation in a distinct subpopulation of nonadherent mature macrophages.

Background

Fourteen-membered ring macrolides have been effective in reducing chronic airway inflammation and also preventing lung injury and fibrosis in bleomycin-challenged mice via anti-inflammatory effects. EM703 is a new derivative of erythromycin (EM) without the bactericidal effects. We investigated the anti-inflammatory and antifibrotic effects of EM703 in an experimental model of bleomycin-induced lung injury and subsequent fibrosis in mice.

Methods

Seven-week-old male ICR mice were used. All experiments used eight mice/group, unless otherwise noted in the figure legends. Bleomycin was administered intravenously to the mice on day 0. EM703 was orally administered daily to mice. All groups were examined for cell populations in the bronchoalveolar lavage (BAL) fluid and for induction of messenger RNA (mRNA) of Smad3 and Smad4 in the lung tissues by reverse transcriptase (RT)-polymerase chainreaction (PCR) on day 7. Fibroblastic foci were assessed histologically, and the hydroxyproline content was chemically determined in the lung tissues on day 28. We performed assay of proliferation and soluble collagen production, and examined the induction of mRNA of Smad3 and Smad4 by RT-PCR in murine lung fibroblast cell line MLg2908. We also examined Smad3, Smad4 and phosphorylated Smad2/3 (p-Smad2/3) protein assay by western blotting in MLg2908.

Results

Bleomycin-induced lung fibrosis, and the infiltration of macrophages and neutrophils into the airspace were inhibited by EM703. The expression of Smad3 and Smad4 mRNA was clearly attenuated by bleomycin, but was recovered by EM703. EM703 also inhibited fibroblast proliferation and the collagen production in lung fibroblasts induced by Transforming growth factor-beta (TGF-β). The expression of Smad3 and Smad4 mRNA in murine lung fibroblasts disappeared due to TGF-β, but was recovered by EM703. EM703 inhibited the expression of p-Smad2/3 and Smad4 protein in murine lung fibroblasts induced by TGF-β.

Conclusion

These findings suggest that EM703 improves bleomycin-induced pulmonary fibrosis in mice by actions of anti-inflammation and regulation of TGF-β signaling in lung fibroblasts.

Dialysis dependency is one of the leading causes of morbidity and mortality in the world, and once end-stage renal disease develops, it cannot be reversed by currently available therapy. Although administration of large doses of bone morphogenetic protein–7 (BMP-7) has been shown to repair established renal injury and improve renal function, the pathophysiological role of endogenous BMP-7 and regulatory mechanism of its activities remain elusive. Here we show that the product of uterine sensitization-associated gene–1 (USAG1), a novel BMP antagonist abundantly expressed in the kidney, is the central negative regulator of BMP function in the kidney and that mice lacking USAG-1 (USAG1–/– mice) are resistant to renal injury. USAG1–/– mice exhibited prolonged survival and preserved renal function in acute and chronic renal injury models. Renal BMP signaling, assessed by phosphorylation of Smad proteins, was significantly enhanced in USAG1–/– mice with renal injury, indicating that the preservation of renal function is attributable to enhancement of endogenous BMP signaling. Furthermore, the administration of neutralizing antibody against BMP-7 abolished renoprotection in USAG1–/– mice, indicating that USAG-1 plays a critical role in the modulation of renoprotective action of BMP and that inhibition of USAG-1 is a promising means of development of novel treatment for renal diseases.

Transcription factor GATA-2 is essential for definitive hematopoiesis, which developmentally emerges from the para-aortic splanchnopleura (P-Sp). The expression of a green fluorescent protein (GFP) reporter placed under the control of a 3.1-kbp Gata2 gene regulatory domain 5′ to the distal first exon (IS) mirrored that of the endogenous Gata2 gene within the P-Sp and yolk sac (YS) blood islands of embryonic day (E) 9.5 murine embryos. The P-Sp- and YS-derived GFP+ fraction of flow-sorted cells dissociated from E9.5 transgenic embryos contained far more CD34+/c-Kit+ cells than the GFP− fraction did. When cultured in vitro, the P-Sp GFP+ cells generated both immature hematopoietic and endothelial cell clusters. Detailed transgenic mouse reporter expression analyses demonstrate that five GATA motifs within the 3.1-kbp Gata2 early hematopoietic regulatory domain (G2-EHRD) were essential for GFP expression within the dorsal aortic wall, where hemangioblasts, the earliest precursors possessing both hematopoietic and vascular developmental potential, are thought to reside. These results thus show that the Gata2 gene IS promoter is regulated by a GATA factor(s) and selectively marks putative hematopoietic/endothelial precursor cells within the P-Sp.

MafA is a transcription factor that binds to the promoter in the insulin gene and has been postulated to regulate insulin transcription in response to serum glucose levels, but there is no current in vivo evidence to support this hypothesis. To analyze the role of MafA in insulin transcription and glucose homeostasis in vivo, we generated MafA-deficient mice. Here we report that MafA mutant mice display intolerance to glucose and develop diabetes mellitus. Detailed analyses revealed that glucose-, arginine-, or KCl-stimulated insulin secretion from pancreatic β cells is severely impaired, although insulin content per se is not significantly affected. MafA-deficient mice also display age-dependent pancreatic islet abnormalities. Further analysis revealed that insulin 1, insulin 2, Pdx1, Beta2, and Glut-2 transcripts are diminished in MafA-deficient mice. These results show that MafA is a key regulator of glucose-stimulated insulin secretion in vivo.