First description of the complete human xylosyltransferase-I promoter region

Abstract

BackgroundHuman xylosyltransferase-I (XT-I) catalyzes the rate-limiting step in proteoglycan glycosylation. An increase in XYLT1 mRNA expression and serum XT activity is associated with diseases characterized by abnormal extracellular matrix accumulation like, for instance, fibrosis. Nevertheless, physiological and pathological mechanisms of transcriptional XT regulation remain elusive.ResultsTo elucidate whether promoter variations might affect the naturally occurring variability in serum XT activity, a complete sequence analysis of the XYLT1 promoter was performed in genomic DNA of healthy blood donors. Based on promoter amplification by a specialized PCR technique, sequence analysis revealed a fragment of 238 bp, termed XYLT1238*, which has never been described in the human XYLT1 reference sequence so far. In silico characterization of this unconsidered fragment depicted an evolutionary conservation between sequences of Homo sapiens and Pan troglodytes (chimpanzee) or Mus musculus (mouse), respectively. Promoter activity studies indicated that XYLT1238* harbors various transcription factor binding sites affecting basal XYLT1 expression and inducibility by transforming growth factor-β1, the key fibrotic mediator.A microsatellite and two single nucleotide variants (SNV), c.-403C>T and c.-1088C>A, were identified and genotyped in 100 healthy blood donors. Construct associated changes in XYLT1 promoter activity were detected for several sequence variants, whereas serum XT activity was only marginally affected.ConclusionsOur findings describe for the first time the entire XYLT1 promoter sequence and provide new insights into transcriptional regulation of XT-I. Future studies should analyze the impact of regulatory XYLT1 promoter variations on XT-associated diseases.

Activin A-Mediated Regulation of XT-I in Human Skin Fibroblasts

“…In contrast to the promoter region of XYLT1, the XYLT2 promoter does not exhibit a binding site for the AP1 transcription factor, which plays a considerable role in the regulation of XYLT1 gene by JNK and p38 [43], explaining why basal XYLT2 expression was not influenced by the usage of p38 and JNK inhibitors in this study. While the XYLT1 promoter exhibits numerous transcription-binding sites for EGR1, which was previously shown to response to non-canonical, Smad3-independent TGFβ pathway MEK/ERK in SScF [60], the main transcription factors substantially involved in XYLT2 promoter regulation are SP1/3 [29,30,62]. These findings are consistent with previously published data demonstrating dose-dependent inhibition of SMAD3 promoter activity by ERK inhibitor UO126 that correlates with the inhibition of SP1/SP3 function by the same inhibitor [63].…”

“…The correlation of mRNA expression and enzyme activity increase regarding XT-I has been shown in this and numerous other studies using TGFβ1-treated human dermal and cardiac fibroblasts [26,27,[45][46][47]. Furthermore, we observed, in consistency with previous results in NHDF [25], that the extracellular enzyme activity of untreated cells increased over time due to the protein accumulation in the cell supernatant, while intracellular XT activity remains constant over the test period of 120 h. These findings support the hypothesis that two regulatory mechanisms exist to control enzyme activity in NHDF: One at the transcriptional stage and another operating post-translationally, shedding the Golgi-resident, constitutive active enzyme from the membrane for release to the extracellular space controlling the rate of PG biosynthesis by reducing the cellular enzyme amount [24,29]. In contrast to TGFβ1, activin A treatment of NHDF results in a weaker XYLT1 mRNA expression increase, which can be explained by the autocrine TGFβ1 signalling, which is responsible for sustaining or amplifying the fibrotic responses in the pathogenesis of SSc [7].…”

“…Former sequence analysis of the human XYLT1 promoter region revealed several downstream transcription factors of the EGR-, SP1-or KLF-family to regulate XYLT1 mRNA expression [28,29,43]. Giving the missing link between promoter analysis, identified transcription factors and upstream cellular signalling mediating XYLT1 expression in response to TGFβ cytokine stimulation of ALK receptors, we found a novel signalling axis involving MAPK and Smad proteins in activin A-mediated XT-I regulation in primary NHDF cells.…”

“…It might therefore be plausible that the slight increase in basal XYLT1 expression observed in the Smad3-inhibited fibroblast cells of this study were due to this SNV. As former studies did not reveal any differences in serum enzyme activity in these blood donors harbouring the appropriate XYLT1 promoter SNV c.-1088C>A [29], the corresponding XYLT1 promoter SNV in this cells was therefore not considered further by sequencing for dividing into subgroups.…”

“…Former studies using activin A and a human neuroblastoma cell line demonstrated that the expression of some TGFβ superfamily target genes induced by ALK4-activin A did not require promoter bindings of SMAD2/3 [10]. By taking into consideration that of the XYLT1 promoter does not provide the according Smad binding site [29], but Smad2 linker phosphorylation was shown to enhance the nuclear localisation of Smad2 proteins [55], we presume that a potential activin A-ALK4 signalling pathway in NHDF is transduced by the MAPK JNK, p38 and ERK pathways and potential phosphorylation of the Smad2 linker region, promoting the nuclear entrance and favoured binding of Smad2 to the former identified transcription factors AP1 and SP1/3, known to increase XYLT1 expression [28,29,[58][59][60][61].…”