RESUMEN
Fibroblast growth factor 23 (FGF23) functions in an endocrine fashion and requires α-Klotho to exert its effects on the target organs. We have recently demonstrated that the human placenta also expresses α-Klotho, which led us to hypothesize that FGF23 may exert effects on the placenta. Immunohistochemical analysis demonstrated the expression of FGF receptor 1 (FGFR1) as well as that of α-Klotho in the feto-maternal interface of both mouse and human normal-term placentas, which suggested that these areas might be receptive to FGF23. Therefore, we next investigated whether FGF23 has some roles in the placenta using Hyp mice with high levels of circulating FGF23. Hyp and wild-type (WT) females were mated with WT males, and the mothers and their male fetuses were analyzed. FGF23 levels in Hyp mothers were elevated. FGF23 levels were about 20-fold higher in Hyp fetuses than in Hyp mothers, whereas WT fetuses from Hyp mothers exhibited low levels of FGF23, as did fetuses from WT mothers. We analyzed the placental gene expression and found that the expression of Cyp24a1 encoding 25OHD-24-hydroxylase, a target gene for FGF23 in the kidney, was increased in the placentas of fetuses from Hyp mothers compared with fetuses from WT mothers. In an organ culture of WT placentas, treatment with plasma from Hyp mothers markedly increased the expression of Cyp24a1, which was abolished by the simultaneous addition of anti-FGF23 neutralizing antibody. The direct injection of recombinant FGF23 into WT placentas induced the expression of Cyp24a1. The increase in the placental expression of Cyp24a1 in fetuses from Hyp mothers resulted in decreased plasma 25-hydroxyvitamin D levels. These results suggest that increased levels of circulating FGF23 in pathological conditions such as Hyp mice exerts direct effects on the placenta and affects fetal vitamin D metabolism via the regulation of Cyp24a1 expression.
Asunto(s)
Raquitismo Hipofosfatémico Familiar/sangre , Factores de Crecimiento de Fibroblastos/sangre , Placenta/metabolismo , Vitamina D/metabolismo , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/genética , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/metabolismo , Animales , Anticuerpos Neutralizantes/farmacología , Calcio/sangre , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Raquitismo Hipofosfatémico Familiar/genética , Femenino , Feto/efectos de los fármacos , Feto/metabolismo , Factor-23 de Crecimiento de Fibroblastos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Glucuronidasa/genética , Glucuronidasa/metabolismo , Humanos , Riñón/embriología , Riñón/metabolismo , Proteínas Klotho , Masculino , Intercambio Materno-Fetal/efectos de los fármacos , Intercambio Materno-Fetal/genética , Ratones , Minerales/metabolismo , Técnicas de Cultivo de Órganos , Fosfatos/sangre , Placenta/efectos de los fármacos , Embarazo , Receptores de Factores de Crecimiento de Fibroblastos/genética , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factores de Tiempo , Vitamina D/sangre , Vitamina D3 24-Hidroxilasa/genética , Vitamina D3 24-Hidroxilasa/metabolismoRESUMEN
Intrauterine infection is still a common trigger of preterm delivery (PTD) and also a determinant risk factor for the subsequent development of neurodevelopmental abnormalities in neonates. In this study, we examined the expressional pattern of various inflammatory cytokines such as interleukin-1ß (IL-1ß), IL-6 and tumor necrosis factor-α (TNF-α) in placentae complicated with severe chorioamnionitis (CAM) and found that IL-6 is mainly expressed in macrophages in villous mesenchyme by immunohistochemical analysis with anti-CD-68 antibody. Using an experimental lipopolysaccharide (LPS)-induced PTD model, the therapeutic potential of targeting this cytokine was investigated. Anti-IL-6 receptor antibody (MR16-1) was delivered 6 h before LPS treatment. Mice in the MR16-1 group had a significantly lower rate of PTD (17%) than in the controls (53%, P = 0.026). As a result, MR16-1 treatment significantly prolonged the gestational period (control; 18.4 ± 1.7d, MR16-1; 19.8 ± 1.5d, P = 0.007) without any apparent adverse events on the mice and their pups. In primary human amniotic epithelial cells, pretreatment with a humanized anti-human IL-6 receptor antibody, tocilizumab, significantly inhibited the production of prostaglandin E2 induced by IL-6. In conclusion, IL-6 was strongly expressed mainly in macrophages in villous mesenchyme in placentae complicated with CAM. Anti-IL-6R antibody significantly decreased the rate of PTD in LPS-induced inflammatory model in mice, and inhibited PGE2 production from human primary amniotic epithelial cells. Targeting IL-6 signaling could be a promising option for the prevention of PTD and needs to be further explored for future clinical application.
Asunto(s)
Anticuerpos/farmacología , Inflamación/complicaciones , Terapia Molecular Dirigida , Nacimiento Prematuro/prevención & control , Receptores de Interleucina-6/antagonistas & inhibidores , Animales , Anticuerpos/uso terapéutico , Estudios de Casos y Controles , Células Cultivadas , Vellosidades Coriónicas/efectos de los fármacos , Vellosidades Coriónicas/metabolismo , Femenino , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Embarazo , Nacimiento Prematuro/etiología , Receptores de Interleucina-6/inmunologíaRESUMEN
AIM: The molecular mechanisms by which hepatocyte nuclear factor (HNF)4α regulates fetal liver development have not been fully elucidated. We screened the downstream molecules of HNF4α during liver development and identified sodium-coupled neutral amino acid transporter (SNAT)4. The aim of this study is to investigate the regulation of SNAT4 by HNF4α and to clarify its roles in differentiating hepatocytes. METHODS: HNF4α was overexpressed in cultured liver buds using adenovirus, and suppression subtractive hybridization screening was performed. Temporal and spatial expression of SNAT4 during liver development was investigated. Regulation of SNAT4 by HNF4α was examined by promoter analyses and electrophoretic mobility shift assays (EMSA). Metabolic labeling and western blotting were carried out using primary hepatoblasts with SNAT4 overexpression. RESULTS: The expression of Slc38a4 encoding SNAT4 showed a marked perinatal increase, and was predominant among system A amino acid transporters. It was first detected in embryonic day 18.5 liver, and found in most hepatocytes after birth. Three alternative first exons were found in the SNAT4 gene. Promoter analyses using approximately 3-kb fragments corresponding to each first exon (AP1, AP2, AP3) revealed that AP1 and AP2 exhibited strong promoter activity in mouse hepatoblasts with endogenous HNF4α. Transactivation of AP2 was upregulated by HNF4α in HeLa cells without endogenous HNF4α. EMSA has demonstrated that HNF4α directly binds to cis-elements in AP2. Overexpression of SNAT4 facilitated amino acid uptake and de novo protein synthesis in primary hepatoblasts. CONCLUSION: SNAT4 functions downstream of HNF4α and plays significant roles in liver development through mechanisms of amino acid uptake and protein synthesis.
RESUMEN
Fibroblast growth factor-23 (FGF23) is well established to play crucial roles in the regulation of phosphate homeostasis. X-linked hypophosphatemic rickets (XLH) is characterized by impaired mineralization and growth retardation associated with elevated circulating FGF23 levels. Administration of phosphate and calcitriol is effective in improving growth retardation, but is not sufficient to fully reverse impaired growth, suggesting the existence of a disease-specific mechanism in the development of growth retardation in addition to dysregulated phosphate metabolism. However, the precise mechanisms of growth retardation in XLH remain elusive. Here, we postulated that FGF23 suppressed chondrocyte proliferation in the presence of soluble α-Klotho (sKL). In vitro and ex vivo studies revealed that FGF23 formed a protein complex with sKL through KL1 internal repeat and suppressed the linear growth of metatarsals in the presence of sKL, which was antagonized by co-incubation with neutralizing antibodies against FGF23 or by knocking-down FGFR3 expression. Additionally, FGF23 binding to FGFR3 was enhanced in the presence of sKL. Histologically, the length of the proliferating zone was diminished and was associated with decreased chondrocyte proliferation. FGF23/sKL suppressed Indian hedgehog (Ihh) expression and administration of Ihh protein partially rescued the suppressive effect of FGF23/sKL on metatarsal growth. Intraperitoneal administration of sKL in Hyp mice, a murine model for XLH, caused a decrease in the length of the proliferating zone associated with decreased chondrocyte proliferation without altering circulating phosphate levels. These findings suggest that suppression of chondrocyte proliferation by FGF23 could have a causative role in the development of growth retardation in XLH.
Asunto(s)
Condrocitos/citología , Raquitismo Hipofosfatémico Familiar/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Enfermedades Genéticas Ligadas al Cromosoma X , Receptores de Superficie Celular/metabolismo , Animales , Línea Celular , Proliferación Celular , Condrogénesis , Modelos Animales de Enfermedad , Factor-23 de Crecimiento de Fibroblastos , Regulación de la Expresión Génica , Glucuronidasa , Humanos , Técnicas In Vitro , Proteínas Klotho , Ratones , Ratones Endogámicos C57BL , Mutación , Proteínas Recombinantes/química , Transducción de SeñalRESUMEN
Ureaplasma spp. is detected in the urogenital tract, including the vagina, cervix, chorioamnion, and placenta. Their colonization is associated with histologic chorioamnionitis (CAM), often observed in placentas from preterm delivery. We isolated Ureaplasma spp. from 63 preterm placentas among 151 specimens, which were delivered at <32 wk of gestation. Of the 63 placentas, 52 (83%) revealed CAM in cultures positive for Ureaplasma spp., however, CAM was observed only in 30% (26/88) of cultures negative for Ureaplasma spp. (p < 0.01). Colonization by Ureaplasma spp. was an independent risk factor for CAM (OR, 11.27; 95% CI, 5.09-24.98). Characteristic neutrophil infiltration was observed in the amnion and subchorion (bistratified pattern) in cultures positive for Ureaplasma spp. FISH analysis of CAM placenta with male infant pregnancy indicated that bistratified infiltrated neutrophils showed the XX karyotype and umbilical vein infiltrated neutrophils showed XY karyotype. The distribution of sulfoglycolipid, the receptor of Ureaplasma spp., was mainly detected in the amnion. Ureaplasmal urease D protein and ureB gene were both detected in the amnion, indicating direct colonization by Ureaplasma spp.
Asunto(s)
Corioamnionitis/microbiología , Placenta/microbiología , Nacimiento Prematuro/microbiología , Infecciones por Ureaplasma/microbiología , Ureaplasma/aislamiento & purificación , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Estudios de Casos y Controles , Corioamnionitis/genética , Corioamnionitis/inmunología , Cromosomas Humanos X , Cromosomas Humanos Y , Femenino , Edad Gestacional , Glucolípidos/metabolismo , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Modelos Logísticos , Masculino , Infiltración Neutrófila , Oportunidad Relativa , Placenta/inmunología , Reacción en Cadena de la Polimerasa , Embarazo , Nacimiento Prematuro/genética , Nacimiento Prematuro/inmunología , Medición de Riesgo , Factores de Riesgo , Ureaplasma/genética , Ureaplasma/metabolismo , Infecciones por Ureaplasma/complicaciones , Infecciones por Ureaplasma/genética , Infecciones por Ureaplasma/inmunología , Ureasa/genética , Ureasa/metabolismoRESUMEN
Intrauterine infection is associated with chorioamnionitis (CAM), which can lead to preterm delivery. We previously reported that the levels of IgM and the incidence of CAM were elevated in preterm infants with neonatal pulmonary emphysema. The pathogen and target of this IgM remain unclear. By using Western blot and amino acid sequences, we have determined one of the target proteins: annexin A2. Immunohistochemical analysis showed that annexin A2 was expressed at fetal chorion and amnion membranes. Among very low birth weight (VLBW) infants with hyper-IgM (> or = 30 mg/dL), 58.8% showed a high titer against annexin A2 (more than x 16), which accounted for about 20%-40% of the total IgM. Anti-annexin A2 IgM antibody inhibited plasmin generation. Furthermore, the median of anti-annexin A2 IgM titer from preterm infants who were delivered with high-grade (grade III) CAM was significantly higher than those from preterm infants without CAM (p = 0.011) and with low-grade CAM (grade I and II) (p = 0.010). Here, we indicate the fetal autoimmunoreactivity against the fetomaternal interface in preterm infants.