Transcriptional regulation of FRZB in chondrocytes by Osterix and Msx2.

INTRODUCTION: Osteoarthritis is a common joint disease that causes destruction of articular cartilage and severe inflammation surrounding knee and hip joints. However, to date, effective therapeutic reagents for osteoarthritis have not been developed because the underlying molecular mechanisms are complex. Recent genetic findings suggest that a Wnt antagonist, frizzled-related protein B (FRZB), is a potential therapeutic target for osteoarthritis. Therefore, this study aimed to examine the transcriptional regulation of FRZB in chondrocytes. MATERIALS AND METHODS: Frzb/FRZB expression was assessed by RT-qPCR analyses in murine articular chondrocytes and SW1353 chondrocyte cell line. Overexpression and knockdown experiments were performed using adenovirus and lentivirus, respectively. Luciferase-reporter and chromatin immunoprecipitation assays were performed for determining transcriptional regulation. Protein-protein interaction was determined by co-immunoprecipitation analysis. RESULTS: Frzb was highly expressed in cartilages, especially within articular chondrocytes. Interleukin-1α markedly reduced Frzb expression in articular chondrocytes in association with cartilage destruction and increases in ADAM metallopeptidase with thrombospondin type 1 motif (Adamts) 4 and Adamts5 expression. Bone morphogenetic protein 2 (BMP2) increased FRZB expression in SW1353 cells through Smad signaling. Osterix and msh homeobox 2 (Msx2), both of which function as downstream transcription factors of BMP2, induced FRZB expression and upregulated its promoter activity. Co-immunoprecipitation results showed a physical interaction between Osterix and Msx2. Knockdown of either Osterix or Msx2 inhibited BMP2-dependent FRZB expression. Chromatin immunoprecipitation indicated a direct association of Osterix and Msx2 with the FRZB gene promoter. CONCLUSION: These results suggest that BMP2 regulates FRZB expression through Osterix and Msx2.

Effects of oxidative stress-induced increases in Zn2+ concentrations in human gingival epithelial cells.

BACKGROUND AND OBJECTIVE: Previous studies have reported that oxidative stress increases intracellular Zn2+ concentrations and induces cytotoxicity. However, no studies have investigated whether oxidative stress induces such changes in periodontal tissue cells. In the present study, we investigated the effect of oxidative stress on intracellular Zn2+ concentration in periodontium constituent cells and its potential relationship with periodontal disease. METHODS: We analyzed changes in intracellular Zn2+ concentrations in human gingival epithelial (epi4) cells treated with hydrogen peroxide (H2 O2 ). The fluorescent probes FluoZin-3 AM and CellTracker Green CMFDA were used to detect intracellular Zn2+ and thiol groups, respectively. Western blot analyses, luciferase reporter assays, and real-time polymerase chain reaction (PCR) analyses were performed to examine the effect of intracellular Zn2+ on epi4 cells. RESULTS: H2 O2 treatment increased intracellular concentrations of Zn2+ in epi4 cells by facilitating the movement of Zn2+ from cellular nonprotein thiols to the cytoplasm and promoting cell membrane permeability to Zn2+ . Furthermore, H2 O2 -induced increases in intracellular Zn2+ activated the p38 cAMP response element-binding protein/mitogen-activated protein kinase (p38 CREB/MAPK) cascade, upregulated nuclear factor kappa B (NF-κB) DNA binding, and increased the expression of inflammatory cytokines and matrix metallopeptidase-9 (MMP-9). CONCLUSION: Increases in intracellular Zn2+ induced by oxidative stress activate signaling pathways involved in inflammation, potentially contributing to the progression of periodontal disease.

A novel heterozygous intronic mutation in POU1F1 is associated with combined pituitary hormone deficiency.

POU class 1 homeobox 1 (POU1F1) regulates pituitary cell-specific gene expression of somatotropes, lactotropes, and thyrotropes. In humans, two POU1F1 isoforms (long and short isoform), which are generated by the alternative use of the splice acceptor site for exon 2, have been identified. To date, more than 30 POU1F1 mutations in patients with combined pituitary hormone deficiency (CPHD) have been described. All POU1F1 variants reported to date affect both the short and long isoforms of the POU1F1 protein; therefore, it is unclear at present whether a decrease in the function of only one of these two isoforms is sufficient for disease onset in humans. Here, we described a sibling case of CPHD carrying a heterozygous mutation in intron 1 of POU1F1. In vitro experiments showed that this mutation resulted in exon 2-skipping of only in the short isoform of POU1F1, while the long isoform remained intact. This result strongly suggests the possibility, for the first time, that isolated mutations in the short isoform of POU1F1 could be sufficient for induction of POU1F1-related CPHD. This finding improves our understanding of the molecular mechanisms, and developmental course associated with mutations in POU1F1.

Gradually increasing ethinyl estradiol for Turner syndrome may produce good final height but not ideal BMD.

Estrogen replacement therapy in Turner syndrome should theoretically mimic the physiology of healthy girls. The objective of this study was to describe final height and bone mineral density (BMD) in a group of 17 Turner syndrome patients (group E) who started their ethinyl estradiol therapy with an ultra-low dosage (1-5 ng/kg/day) from 9.8-13.7 years. The subjects in group E had been treated with GH 0.35 mg/kg/week since the average age of 7.4 years. The 30 subjects in group L, one of the historical groups, were given comparable doses of GH, and conjugated estrogen 0.3125 mg/week ∼0.3125 mg/day was initiated at 12.2-18.7 years. The subjects in group S, the other historical group, were 21 patients who experienced breast development and menarche spontaneously. Final height (height gain < 2 cm/year) in group E was 152.4 ± 3.4 cm and the standard deviation (SD) was 2.02 ± 0.62 for Turner syndrome. The final height in group L was 148.5 ± 3.0 cm with a SD of 1.30 ± 0.55, which was significantly different from the values for group E. The volumetric BMD of group S (0.290 ± 0.026 g/cm3) was significantly different from that of group L or E (0.262 or 0.262 g/cm3 as a mean, respectively). This is the first study of patients with Turner syndrome where estrogen was administered initially in an ultra-low dose and then increased gradually. Our estrogen therapy in group E produced good final height but not ideal BMD.

Fertility preservation in a family with a novel NR5A1 mutation.

The common phenotype of nuclear receptor superfamily 5, group A, member 1 (NR5A1) gene mutations in 46,XY is gonadal dysgenesis without adrenal deficiency. Though the phenotype of gonadal dysgenesis is variable, ranging from complete female to normal male genitalia, an asymptomatic 46,XY male is rare. Preserved fertility has so far been described in only three affected 46,XY males with different mutations, but no functional analysis of these mutations has been performed. Here, we report on male siblings with hypospadias and their asymptomatic father in whom we identified a heterozygous NR5A1 mutation of c.910G>A, p.E304K. Western blotting and subcellular localization revealed no significant difference between the wild type (WT) and E304K. Electrophoretic mobility shift assay experiments showed that E304K abrogated DNA-binding ability. E304K reduced transactivation and had no dominant negative effect. In conclusion, we report on a novel hypomorphic NR5A1 mutation, which may be associated with the phenotype of the family.

Sclerosteosis (craniotubular hyperostosis-syndactyly) with complex hyperphalangy of the index finger.

We report a 4-year-old boy with sclerosteosis associated with severe digital dysostosis. The initial medical consultation was prompted by bilateral, asymmetrical syndactyly of the index and middle fingers. The left index finger had complicated phalangeal anomalies: hyperphalangy (supernumerary phalanx distal to the middle phalanx) and hypoplasia with bracket epiphyses of the proximal and middle phalanges. Development of facial nerve palsy, hearing impairment and generalized osteosclerosis had occurred between 3 years and 4 years of age, with the subsequent identification of a homozygous SOST mutation. Bilateral second and third fingers syndactyly associated with abnormal patterning of the same fingers should be considered prodromal signs of sclerosteosis.

[Bone and Stem Cells. Regulation of chondrocyte differentiation from mesenchymal stem cells].

Chondrocytes are derived from mesenchymal stem cells and play an essential role in endochondral ossification. Transcription factors, Sox9, Runx2, Runx3 and Osterix are critical for endochondral ossification, and regulate differentiation of mesenchymal stem cells into chondrocytes, and proliferation, maturation and apoptosis of chondrocytes. Recent advances in gene cloning approaches utilizing microarray and high-throughput sequencing technologies have revealed functional regulatory mechanisms of these transcription factors and contributed to understanding of molecular mechanisms of complex and harmonious chondrocyte differentiation.

[Modulation of transcriptional regulation during bone and cartilage development and their disease].

Genetic and biochemical studies have identified transcription factors critical and specific for bone and cartilage development. More recent studies revealed the molecular mechanisms how these transcription factors regulate bone and cartilage development. Especially, we appreciate recent advances in molecular function of the complex assembled by these transcription factors and epigenetic regulation of them. Aging, inflammation, biological stress, and disorder of endocrine system induce several bone and/or cartilage diseases by affecting the transcriptional and epigenetic regulation. In this review, we would like to describe the transcriptional and epigenetic regulation during developmental and pathological stages. In addition, we discuss possible application of these information in regeneration of bone and cartilage.

Screening for imprinting disorders in 58 patients with clinically diagnosed idiopathic short stature.

Objectives Imprinted genes have important roles for normal growth and development. Imprinting disorders (IDs) such as Silver-Russell syndrome and Temple syndrome are rare diseases that typically cause short children born small for gestational age (SGA). However, some patients with short stature (SS) caused by IDs were born non-SGA. To date, the contribution of IDs to idiopathic short stature (ISS) has been poorly investigated. The aim of this study was to clarify the contribution of IDs to ISS. Methods We conducted methylation analysis for 10 differentially methylated regions using pyrosequencing to detect known IDs in 58 patients (31 male and 27 female children, height standard deviation score -4.2 to -2.0) carrying a clinical diagnosis of ISS. Results We identified no patient with IDs among these patients with ISS. Conclusions These results indicate that IDs are rare in patients having ISS, and that imprinted genes affect fetal growth more than postnatal growth. Because patients with IDs born non-SGA usually have clinical features characteristic of each ID, in addition to SS, the patients with ISS as a clinical diagnosis may not be associated with IDs. It is unlikely that cases clinically diagnosed with ISS are caused by IDs leading to growth failure.

Relaxin-3 regulates corticotropin-releasing factor gene expression in cultured rat hypothalamic 4B cells.

The ancestral insulin/relaxin peptide superfamily member relaxin-3 is an important regulator of food intake and behaviors related to anxiety and motivation. Relaxin family peptide receptor 1 (RXFP1) and RXFP3 are expressed in the rat hypothalamic paraventricular nucleus (PVN). Corticotropin-releasing factor (CRF) is produced in the PVN in response to stressors and promotes adrenocorticotropic hormone secretion from the anterior pituitary. We hypothesized that relaxin-3 directly regulates Crf expression in the hypothalamus and investigated its effect on Crf expression in cultured hypothalamic 4B cells. Relaxin-3 increased Crf mRNA levels and stimulated Crf promoter activity. Both protein kinase A and C pathways contributed to relaxin-3-induced Crf promoter activity. Rxfp1 and Rxfp3 mRNA and their proteins were expressed in cultured hypothalamic 4B cells. Relaxin-3 decreased Rxfp1 mRNA and protein levels and increased Rxfp3 mRNA and protein levels. These results suggested that the action of relaxin-3 in cultured hypothalamic 4B cells may be regulated through both RXFP1 and RXFP3.

Interaction of LEF1 with TAZ is necessary for the osteoblastogenic activity of Wnt3a.

Canonical Wnt signalling plays an important role in osteoblast differentiation and bone formation. However, the molecular mechanisms by which canonical Wnt signalling exerts its osteoblastogenic effect remain elusive. Here, we investigated the relationship between lymphoid enhancer-binding factor 1 (LEF1) and transcriptional co-activator with PDZ-binding motif (TAZ), both of which are transcriptional regulators that mediate canonical Wnt signalling during osteoblast differentiation. Reporter assay and co-immunoprecipitation experiments revealed functional and physical interaction between LEF1 and TAZ. Overexpression of dominant-negative forms of either LEF1 or TAZ markedly inhibited Wnt3a-dependent osteoblast differentiation. Moreover, we found that LEF1 and TAZ formed a transcriptional complex with runt-related transcription factor 2 (Runx2) and that inhibition of LEF1 or TAZ by their dominant-negative forms dramatically suppressed the osteoblastogenic activity of Ruxn2. Additionally, Wnt3a enhanced osteoblast differentiation induced by bone morphogenetic protein 2 (BMP2), which stimulates osteoblast differentiation by regulating Runx2. Collectively, these findings suggest that interaction between LEF1 and TAZ is crucial for the osteoblastogenic activity of Wnt3a and that LEF1 and TAZ contribute to the cooperative effect of Wnt3a and BMP2 on osteoblast differentiation through association with Runx2.

Novel histidine-conjugated galactosylated cationic liposomes for efficient hepatocyte-selective gene transfer in human hepatoma HepG2 cells.

To enhance gene transfection to hepatocytes by cationic liposomes, it is necessary to overcome a number of barriers existing in the process from administration to gene expression. Recently we and other group have demonstrated that the escape of plasmid DNA (pDNA)/cationic liposome complexes (lipoplexes) from the endosome to cytoplasm was rate limiting. In this study, to enhance transfection efficiency by promoting the release of lipoplexes from the endosome to cytoplasm, we proposed utilizing the "proton sponge effect". Here, we synthesized a novel pH-sensitive histidine-modified galactosylated cholesterol derivative (Gal-His-C4-Chol), for a more efficient gene delivery to hepatocytes. Liposomes containing Gal-His-C4-Chol showed much greater transfection activity than conventional Gal-C4-Chol liposomes based on a receptor-mediated mechanism in HepG2 cells. Hence, this finding should contribute to the development of gene therapy using cationic liposomes toward their clinical application.

Regulation of gonadotropins by urocortin 2 in gonadotropic tumor LßT2 cells.

A close interaction has been shown between the hypothalamo-pituitary-gonadal axis and the hypothalamic-pituitary-adrenal axis. Urocortin 2 (Ucn2) has a very high affinity for the corticotropin-releasing factor (CRF) type 2 (CRF2) receptor. Pituitary Ucn2 regulates expression and secretion of gonadotropins in response to stress. The CRF2 receptor in the pituitary contributes to the modulation of gonadotropins. To explore the possible function of Ucn2 and the CRF2 receptor in pituitary gonadotropic tumor cells, we examined the direct regulation of gonadotropins by Ucn2 in a representative pituitary gonadotropic tumor, mouse LßT2 cells. LßT2 cells were found to express CRF1 receptor and CRF2 receptor mRNA. Ucn2 decreased CRF1 receptor mRNA levels, while it increased CRF2 receptor mRNA levels. Ucn2 directly decreased the mRNA levels of both luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in LßT2 cells. Ucn2 also decreased gonadotropin-releasing hormone receptor (GnRHR) mRNA levels. A selective CRF2 receptor antagonist suppressed the Ucn2-induced decreases in LH, FSH, and GnRHR mRNA levels. Ucn2 acts on gonadotrophs expressing the CRF2 receptor, and inhibits the production of gonadotropins in the pituitary gonadotropic tumor cells. (177 words).

Regulation of Cartilage Development and Diseases by Transcription Factors.

Genetic studies and molecular cloning approaches have been successfully used to identify several transcription factors that regulate the numerous stages of cartilage development. Sex-determining region Y (SRY)-box 9 (Sox9) is an essential transcription factor for the initial stage of cartilage development. Sox5 and Sox6 play an important role in the chondrogenic action of Sox9, presumably by defining its cartilage specificity. Several transcription factors have been identified as transcriptional partners for Sox9 during cartilage development. Runt-related transcription factor 2 (Runx2) and Runx3 are necessary for hypertrophy of chondrocytes. CCAAT/enhancer-binding protein ß (C/EBPß) and activating transcription factor 4 (ATF4) function as co-activators for Runx2 during hypertrophy of chondrocytes. In addition, myocyte-enhancer factor 2C (Mef2C) is required for initiation of chondrocyte hypertrophy, presumably by functioning upstream of Runx2. Importantly, the pathogenic roles of several transcription factors in osteoarthritis have been demonstrated based on the similarity of pathological phenomena seen in osteoarthritis with chondrocyte hypertrophy. We discuss the importance of investigating cellular and molecular properties of articular chondrocytes and degradation mechanisms in osteoarthritis, one of the most common cartilage diseases.

Syndromic disorder of sex development due to a novel hemizygous mutation in the carboxyl-terminal domain of ATRX.

Alpha-thalassemia/mental retardation syndrome X-linked (ATRX; OMIM #301040), which is caused by mutations in the ATRX gene, is characterized by alpha-thalassemia, distinct dysmorphic facies, psychomotor development delay and genital abnormalities. Here, we describe a neonatal case of syndromic disorder of sex development, harboring a novel hemizygous mutation, p.Asp2352fs*1 in the carboxyl-terminal domain of ATRX. Our study provides additional evidence that deletion of the carboxyl terminus of ATRX is associated with severe genital anomalies.

Five cases of childhood-onset Graves' disease treated with either surgery or radio-iodine therapy.

There are three major therapeutic options for the treatment of Graves' disease (GD): antithyroid drugs (ATDs), thyroidectomy, and radio-iodine (RAI) therapy. ATDs are the initial treatment option for children. However, some pediatric GD patients who are initially treated with ATDs require other type of treatments later on. We reviewed the medical records of childhood-onset GD cases retrospectively to report the clinical course of patients who received either surgery or RAI therapy subsequent to treatment with ATDs. Childhood-onset GD was successfully managed in five girls with non-ATD treatments at the age of 7-14 yr following an unfavorable outcome of initial ATD treatment. Four cases had surgery and one case was managed with RAI therapy. The reasons for switching to non-ATD treatment included poor compliance, failure to maintain remission, serious adverse events resulting from ATDs, and religious background. In conclusion, surgery and RAI therapy could be good alternative treatment options for children with GD.

Functional Characterization of c.870+3_6delGAGT Splice Site Mutation in NR5A1.

BACKGROUND: To date, more than 100 mutations of NR5A1 have been reported; however, mutations affecting the splice site are rare, with only two reported mutations. OBJECTIVE: To characterize the c.870+3_6delGAGT splice mutation of NR5A1 through molecular analyses. RESULTS: The reverse transcription polymerase chain reaction (RT-PCR) study revealed that c.870+3_6delGAGT resulted in p.A82fs*95. Mutant NR5A1 showed a reduced transactivation on the CYP11A1 and STAR promoters without a dominant negative effect. CONCLUSION: To the best of our knowledge, this is the first report of the NR5A1 splice site mutation, which was proven to be deleterious by the RT-PCR method.

Novel heterozygous mutation in the extracellular domain of FGFR1 associated with Hartsfield syndrome.

Heterozygous kinase domain mutations or homozygous extracellular domain mutations in FGFR1 have been reported to cause Hartsfield syndrome (HS), which is characterized by the triad of holoprosencephaly, ectrodactyly and cleft lip/palate. To date, more than 200 mutations in FGFR1 have been described; however, only 10 HS-associated mutations have been reported thus far. We describe a case of typical HS with hypogonadotropic hypogonadism (HH) harboring a novel heterozygous mutation, p.His253Pro, in the extracellular domain of FGFR1. This is the first report of an HS-associated heterozygous mutation located in the extracellular domain of FGFR1, thus expanding our understanding of the phenotypic features and further developmental course associated with FGFR1 mutations.