RESUMEN
The dopamine transporter (DAT) is a key regulator of dopaminergic neurotransmission. As such, proper regulation of DAT expression is important to maintain homeostasis, and disruption of DAT expression can lead to neurobehavioral dysfunction. Based on genomic features within the promoter of the DAT gene, there is potential for DAT expression to be regulated through epigenetic mechanisms, including DNA methylation and histone acetylation. However, the relative contribution of these mechanisms to DAT expression has not been empirically determined. Using pharmacologic and genetic approaches, we demonstrate that inhibition of DNA methyltransferase (DNMT) activity increased DAT mRNA approximately 1.5-2 fold. This effect was confirmed by siRNA knockdown of DNMT1. Likewise, the histone deacetylase (HDAC) inhibitors valproate and butyrate also increased DAT mRNA expression, but the response was much more robust with expression increasing over tenfold. Genetic knockdown of HDAC1 by siRNA also increased DAT expression, but not to the extent seen with pharmacological inhibition, suggesting additional isoforms of HDAC or other targets may contribute to the observed effect. Together, these data identify the relative contribution of DNMTs and HDACs in regulating expression. These finding may aid in understanding the mechanistic basis for changes in DAT expression in normal and pathophysiological states.
Asunto(s)
Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/genética , Epigénesis Genética , Neuroblastoma/genética , ARN Mensajero/genética , Línea Celular Tumoral , Metilasas de Modificación del ADN/genética , Técnicas de Silenciamiento del Gen , Histona Desacetilasas/genética , Humanos , Neuroblastoma/patología , ARN Interferente Pequeño/genéticaRESUMEN
OBJECTIVE: Progesterone (P(4)) has been clinically shown to prevent the recurrence of preterm birth. The mechanism(s) of action is unclear, but may involve modulation of the immunologic inflammatory response of the lower genital tract. We evaluated the effects of P(4) on interleukin-8 (IL-8) production by vaginal and cervical epithelial cells stimulated with bacterial species that are commonly associated with preterm birth. METHODS: Vaginal and endocervical epithelial cells were incubated with up to 10,000 ng/mL P(4) overnight and stimulated with heat-killed Escherichia coli, Gardnerella vaginalis, or Ureaplasma urealyticum. Concentrations of IL-8 in conditioned medium were quantified by ELISA and viability of the cell cultures was measured by the reduction of a tetrazolium salt. RESULTS: E. coli, G. vaginalis and U. urealyticum-stimulated IL-8 production for both cell lines. P(4) inhibited basal and bacteria-stimulated IL-8 production for vaginal epithelial cells but enhanced IL-8 production by endocervical cells. P(4) reduced the number of viable cells for both cell lines. CONCLUSIONS: P(4) inhibits IL-8 production by vaginal epithelial cells stimulated with pathogens associated with preterm birth, possibly by reducing the number of viable cells or by inhibiting their proliferation. Although P(4) also reduces proliferation of endocervical cells it also increases their production of IL-8.
Asunto(s)
Células Epiteliales/efectos de los fármacos , Interleucina-8/metabolismo , Nacimiento Prematuro/prevención & control , Progesterona/farmacología , Línea Celular , Cuello del Útero/citología , Cuello del Útero/inmunología , Cuello del Útero/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Femenino , Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/inmunología , Humanos , Inmunidad Innata , Nacimiento Prematuro/metabolismo , Nacimiento Prematuro/microbiología , Progesterona/uso terapéutico , Vagina/citología , Vagina/inmunología , Vagina/metabolismoRESUMEN
PROBLEM: Sulfasalazine (SASP) inhibits lipopolysaccharide-induced nuclear-factor kappa B activation and interleukin-8 (IL-8) production by cultured explants of placenta, amnion and choriodecidua. Bacteria-induced IL-8 production in the cervix is a potential mechanism for premature cervical ripening that may lead to preterm birth. Our objective was to determine if SASP inhibits IL-8 production by endocervical cells stimulated with bacterial pathogens associated with preterm birth. METHOD OF STUDY: Human endocervical cells were incubated with 0-1.6 mm SASP and then stimulated with Ureaplasma parvum, Escherichia coli, or Gardnerella vaginalis. Conditioned medium was then harvested and production of IL-8 was quantified by ELISA. Viability of the cells was ascertained at the end of the experiment with the MTT-assay. RESULTS: At the highest concentration tested (1.6 mm), SASP significantly inhibited IL-8 production by cultures stimulated with E. coli (P < 0.001), U. parvum (P < 0.001), and G. vaginalis (P < 0.001). Viability of the cells, however, was significantly reduced by SASP at 0.8 and 1.6 mm in both the presence and absence of bacteria for all experiments. CONCLUSION: Although high concentrations of SASP inhibit IL-8 production by cultures of endocervical cells stimulated with pathogens associated with preterm birth, this effect may be because of toxicity of the antibiotic on the cells.
Asunto(s)
Antiinfecciosos/farmacología , Antiinflamatorios no Esteroideos/farmacología , Cuello del Útero/inmunología , Interleucina-8/antagonistas & inhibidores , Nacimiento Prematuro/inmunología , Sulfasalazina/farmacología , Bacterias/efectos de los fármacos , Bacterias/inmunología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Cuello del Útero/microbiología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/inmunología , Células Epiteliales/microbiología , Femenino , Humanos , Interleucina-1beta/biosíntesis , Interleucina-1beta/efectos de los fármacos , Interleucina-8/biosíntesis , Lipopolisacáridos/inmunología , Monocitos/efectos de los fármacos , Monocitos/inmunología , Nacimiento Prematuro/microbiología , Nacimiento Prematuro/prevención & control , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
PROBLEM: A number of clinical trials have demonstrated that supplemental progesterone (P4) can prevent preterm birth. Although P4 can decrease the production of mediators of inflammation by lipopolysaccharide (LPS)-stimulated macrophages, a majority of infections associated with preterm birth are due to Ureaplasma urealyticum, which does not contain LPS. Therefore, we studied whether P4 could lower the production of proinflammatory cytokines by monocytes stimulated with U. urealyticum. METHOD OF STUDY: Human monocytes (THP-1 cells) were treated with P4 and then stimulated with heat-killed Escherichia coli or U. urealyticum. Cytokine concentrations in the conditioned medium were then measured by ELISA and relative viability of the cells was assessed colorimetrically. The impact of P4 on interleukin (IL)-1beta, tumor necrosis factor-alpha (TNF-alpha) and IL-8 production was assessed by comparing levels across different P4 dosages and organism concentrations. RESULTS: Both organisms increased IL-1alpha, TNF-alpha and IL-8 production in a dose-dependent manner. P4 enhanced the production of IL-1beta and IL-8, but inhibited TNF-alpha production by monocytes stimulated with either organism. CONCLUSION: P4 modulates cytokine production by E. coli and U. urealyticum-stimulated monocytes in a similar manner and is not strictly immunosuppressive. This suggests that P4 does not prevent preterm birth by simply suppressing bacteria-stimulated cytokine production.