RESUMO
Aberrant expression of the human hyaluronan synthase 2 (HAS2) gene has been implicated in the pathology of malignancy, pulmonary arterial hypertension, osteoarthritis, asthma, thyroid dysfunction, and large organ fibrosis. Renal fibrosis is associated with increased cortical synthesis of hyaluronan (HA), an extracellular matrix glycosaminoglycan, and we have shown that HA is a correlate of interstitial fibrosis in vivo. Our previous in vitro data have suggested that both HAS2 transcriptional induction and subsequent HAS2-driven HA synthesis may contribute to kidney fibrosis via phenotypic modulation of the renal proximal tubular epithelial cell (PTC). Post-transcriptional regulation of HAS2 mRNA synthesis by the natural antisense RNA HAS2-AS1 has recently been described in osteosarcoma cells, but the antisense transcript was not detected in kidney. In this study, PTC stimulation with IL-1ß or TGF-ß1 induced coordinated temporal profiles of HAS2-AS1 and HAS2 transcription. Constitutive activity of the putative HAS2-AS1 promoter was demonstrated, and transcription factor-binding sequence motifs were identified. Knockdown of Sp1/Sp3 expression by siRNA blunted IL-1ß induction of both HAS2-AS1 and HAS2, and Smad2/Smad3 knockdown similarly attenuated TGF-ß1 stimulation. Inhibition of IL-1ß-stimulated HAS2-AS1 RNA induction using HAS2-AS1-specific siRNAs also suppressed up-regulation of HAS2 mRNA transcription. The thermodynamic feasibility of HAS2-AS1/HAS2 heterodimer formation was demonstrated in silico, and locus-specific cytoplasmic double-stranded RNA was detected in vitro. In summary, our data show that transcriptional induction of HAS2-AS1 and HAS2 occurs simultaneously in PTCs and suggest that transcription of the antisense RNA stabilizes or augments HAS2 mRNA expression in these cells via RNA/mRNA heteroduplex formation.
Assuntos
Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Glucuronosiltransferase/biossíntese , Túbulos Renais Proximais/metabolismo , RNA Antissenso/biossíntese , Transcrição Gênica , Linhagem Celular Tumoral , Células Epiteliais/patologia , Fibrose , Técnicas de Silenciamento de Genes , Glucuronosiltransferase/genética , Humanos , Hialuronan Sintases , Ácido Hialurônico/biossíntese , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Nefropatias/genética , Nefropatias/metabolismo , Nefropatias/patologia , Túbulos Renais Proximais/patologia , Ácidos Nucleicos Heteroduplexes/biossíntese , Ácidos Nucleicos Heteroduplexes/genética , RNA Antissenso/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Elementos de Resposta/genética , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad3/genética , Proteína Smad3/metabolismo , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp3/genética , Fator de Transcrição Sp3/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
The ubiquitous mammalian extracellular matrix glycosaminoglycan hyaluronan (HA) plays a pivotal role in the regulation of cell phenotype in fibrosis and scarring. Transforming growth factor-beta 1 (TGF-ß1) and interleukin-1 beta (IL-1ß) up-regulate hyaluronan synthase (HAS) 1 and HAS2 in dermal fibroblasts and renal proximal tubular epithelial cells, and subsequent HA synthesis regulates cell phenotype. In the present study, we investigated the mechanism of HAS1 transcriptional up-regulation in response to these cytokines. We used 5'-rapid amplification of cDNA ends analysis to identify the 5' end of HAS1 transcripts, resulting in an increase of 26 nucleotides to the HAS1 exon 1 sequence of reference sequence NM_001523. Constitutive luciferase activity of upstream DNA sequences was shown in luciferase reporter assays, but our reporter vector signals were refractory to the addition of TGF-ß1 and IL-1ß. Using siRNAs to knockdown transcription factor mRNAs, we showed that TGF-ß1 up-regulation of HAS1 transcription was mediated via Smad3 but not Smad2, while HAS1 induction by IL-1ï ß was Sp3, not Sp1, dependent.