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Evidence of calcium-dependent pathway in the regulation of human ßbeta;1,3-glucuronosyltransferase-1 (GlcAT-I) gene expression: a key enzyme in proteoglycan synthesis

UMR 7561 CNRS-Université Henri Poincaré Nancy 1, Faculté de Médecine, Vanduvre-lès-Nancy, France

¹Correspondence: UMR CNRS 7561-Université Henri Poincaré Nancy 1, Faculté de Médecine, BP 184, Vanduvre-lès-Nancy 54505, France, E-mail: ouzzine{at}medecine.uhp-nancy.fr

SPECIFIC AIMS

Heparan-and chondroitin-sulfate glycosaminoglycan (GAG) chains of proteoglycans (PGs) are important regulators in a wide range of biological events such as matrix deposition, intracellular signaling, morphogenesis, cell growth, and migration. Although their implication in several disease processes, including arthropathies, Alzheimer’s disease, and cancer, has been established, little is known about the molecular mechanisms mediating the regulation of GAG biosynthesis. Among the glycosyltransferases involved in the GAG synthesis pathway, beta 1,3-glucuronosyltransferase-I (GlcAT-I) is responsible for the completion of GAG-protein linkage tetrasaccharide, a key step prior to polymerization of chondroitin- and heparan-sulfate chains. While the importance of GlcAT-I is established, little is known about the molecular mechanisms underlying its regulation. Previous studies showed that intracellular calcium concentration modulates PG synthesis in a variety of cells. The aims of this study were to 1) evaluate whether intracellular calcium regulates GlcAT-I gene expression and 2) characterize GlcAT-I promoter and delineate the elements and transcription factors as well as signaling pathway involved in calcium-induced response.

PRINCIPAL FINDINGS

1. GlcAT-I gene expression is induced by elevation of intracellular calcium
To determine whether calcium-dependent signaling can regulate GlcAT-I gene expression, HeLa cells were treated or not with the calcium ionophore ionomycin or with the intracellular calcium chelator BAPTA-AM, and expression levels of GlcAT-I mRNA were analyzed by real-time polymerase chain reaction (PCR). The results showed that GlcAT-I mRNA level was increased by 30% after treatment with ionmycin (1 µM). In contrast, treatment of the cells with BAPTA-AM (10 µM) resulted in a decrease of GlcAT-I mRNA level of 20%. Evaluation of GAG synthesis using 4-methylumbelliferyl-ßbeta;-D-xyloside as an exogenous primer for GAG chain synthesis indicated that ionomycin treatment of HeLa cells led to an increase of 35% in GAG synthesis, whereas treatment with BAPTA-AM reduced GAG synthesis by 29%. These results indicated that calcium-dependent signals modulate both GlcAT-I gene expression and PG GAG chains synthesis.

2. Elevation of intracellular calcium induces GlcAT-I promoter activity
To investigate further the molecular mechanisms whereby intracellular calcium concentration regulates GlcAT-I gene expression, a 2911 bp region of the GlcAT-I promoter was isolated by Genome Walk technique. The –2911/+29 promoter construct (pGL-2911) was transiently transfected into HeLa cells with a control Renilla luciferase vector and exposed to ionomycin (1 µM). Luciferase assays indicated that transcriptional activity of the promoter was enhanced by 3-fold after treatment with ionomycin. Similarly, a truncation promoter construct –302/+29 (pGL-302) was responsive to ionomycin and exhibited a similar level of induction as observed for the full-length promoter construct pGL-2911. Furthermore, treatment with BAPTA-AM significantly reduced basal promoter activity and completely suppressed the induction by ionomycin. These results suggested that intracellular calcium regulates GlcAT-I promoter activity and indicated that the elements involved in calcium inducibility lie within the –302/+29 bp region.

3. Sp1 is involved in the intracellular calcium modulation of GlcAT-I promoter activity
The –302 bp sequence of the proximal promoter contains two Sp1 and three Ets consensus sequences. To investigate the implication of Sp1 in basal and calcium-induced GlcAT-I promoter activity, we used bisanthracycline (WP631), a potent inhibitor of Sp1 transcription factor. WP631 (1 µM) significantly inhibited (50%) basal promoter activity and completely suppressed the stimulatory effect of ionomycin (Fig. 1 A). Moreover, cotransfection of the –302/+29 promoter construct with pSG-Sp1 vector expressing Sp1 transcription factor enhanced by 2-fold the transcriptional activity of the promoter, suggesting that Sp1 transactivates GlcAT-I promoter (Fig. 1B ).

View larger version (28K): [in this window] [in a new window]   Figure 1. Sp1 transcription factor regulates basal and ionomycin-induced GlcAT-I promoter activity. A) HeLa cells were transfected with pGL-302 reporter construct, then treated or not with 1 µM ionomycin in the presence or absence of 1 µM WP 631. Four hours later, cell extracts were prepared, the Firefly luciferase activity was measured and normalized to Renilla luciferase activity. Activity values are mean ±SD of three independent duplicate assays. B) HeLa cells were cotransfected with pGL-302 reporter construct and Sp1 expressing vector pSG-Sp1 or empty vector pSG5 (control). At 24 h post-transfection, cell extracts were assayed for luciferase activity. C) HeLa cells were transfected with pGL-302 construct containing mutated Sp1A (mSp1A), Sp1B (mSp1B), Sp1C (mSp1C), or both Sp1A and Sp1B (mSp1A/B), respectively, then treated or not with 1 µM ionomycin. Four hours after treatment, cell extracts were assayed for luciferase activity. D) For EMSA and supershift assays, 33P-labeled probe from –73 to –46 was used. Lane 1, probe alone; lane 2, probe plus 10 µg of nuclear proteins; lane 3, same as lane 2 plus 100-fold molar excess of unlabeled oligonucleotide; lane 4, same as lane 2 plus 100-fold molar excess of unlabeled Sp1A-mutated oligonucleotide; lane 5, same as lane 2 plus 2 µg of anti-Sp1 Ab; lane 6, same as lane 2 plus 2 µg of anti-Sp3 Ab. The position of specific complex band is marked by an arrow and the supershifted band by an arrowhead. E) For pulldown assay of ionomycin effect on Sp1 DNA binding, 50 mg of nuclear extracts from cells treated or not with ionomycin (1 µM) for 4 h were incubated with biotinylated Sp1A-probe (oligonucleotide –73 to –46) or unrelated probe (control). Sp1A probe-protein complex was then precipitated by addition of streptavidin-agarose beads. The level of Sp1 protein bound to the probe was determined by Western blot using an anti-Sp1 Ab. Equal amounts of nuclear proteins in the different samples were confirmed by using anti-actin antibodies.

To evaluate the individual contribution of Sp1 binding sites, site-directed mutagenesis was used to disrupt each site. Mutation of Sp1 binding sites at positions –33/–25 (mSp1B) and –6/+4 (mSp1C) did not significantly affect the promoter activity (Fig. 1C ), whereas mutation of the Sp1 binding site at position –65/–56 (mSp1A) reduced promoter activity by 52% as well as ionomycin induction by 55% (Fig. 1C ), suggesting the critical role of Sp1A in both basal and calcium-induced GlcAT-I promoter activity. Mutation of both Sp1A and Sp1B (mSp1A/B) abolished the effect of ionomycin (Fig. 1C ).

4. Elevation of intracellular calcium enhanced Sp1 binding to Sp1A consensus sequence
To determine the nature of the transcription factor that binds to Sp1A sequence, EMSA were performed with oligonucleotide corresponding to the region –73 to –46 of the wild-type (WT) promoter encompassing the Sp1A core sequence in the presence of antibodies to Sp1 or its closely relative homologue Sp3 proteins. The results showed a gel retardation of the probe when incubated with nuclear extract (Fig. 1D ). Furthermore, the addition of anti-Sp1 antibody (Ab) produced a supershifted band whereas addition of anti-Sp3 Ab failed to affect the migration of the complex, indicating that Sp1 protein binds to the Sp1A site on the GlcAT-I promoter (Fig. 1D ).

To evaluate the influence of ionomycin treatment on the binding of Sp1 transcription factor to the promoter, we performed a pulldown assay using biotinylated Sp1A probe with nuclear extracts prepared from HeLa cells treated with ionomycin and an anti-Sp1 Ab. The results revealed an increased Sp1 binding after ionomycin treatment (Fig. 1E ), suggesting that intracellular calcium concentration was able to enhance the relative amounts of Sp1 bound to the Sp1A site of GlcAT-I promoter.

5. Calcium activation of GlcAT-I promoter is mediated by ERK MAP kinase
We next studied the signaling pathway by which ionomycin induced GlcAT-I promoter activity. It has been shown that Sp1 phosphorylation by ERK resulted in an enhancement of its DNA binding activity. We thus analyzed the phosphorylation of ERK by phospho-specific antibodies. We found that ionomycin-induced phosphorylation of ERK, which reached a maximum at 5 min and decreased at 60 min after ionomycin stimulation (Fig. 2 A). To determine whether MEK1/2 activity was necessary for ionomycin activation of the promoter, we analyzed the effect of a specific MEK inhibitor PD98059 on GlcAT-I promoter activity. The data showed that pretreatment with 30 µM PD98059 inhibited ERK phosphorylation mediated by ionomycin (Fig. 2B ). In addition, this treatment decreased both basal and ionomycin-induced GlcAT-I promoter activity by 42% and 57%, respectively (Fig. 2C ). Altogether, these data suggest that intracellular calcium induction of GlcAT-I promoter activity is dependent on the MEK/ERK cascade.

View larger version (21K): [in this window] [in a new window]   Figure 2. Ionomycin stimulation of GlcAT-I promoter is dependent on the activation of the MAPK pathway. A) Cells were treated with 1 µM ionomycin for 0–120 min and phosphorylation of ERK was monitored by immunoblot using antiphospho-ERK1/2 antibodies. Equal expression of ERK1/2 in the different samples was confirmed using anti-ERK1/2 antibodies. B) Cells were pretreated or not with PD98059 (30 µM) 30 min before ionomycin exposure. Five minutes later, cell extracts were analyzed by immunoblotting using antiphospho-ERK1/2 and anti-ERK1/2 antibodies. C) Cells were transfected with pGL-302 construct, pretreated or not with PD98059 (30 µM) 30 min, then treated or not with 1 µM ionomycin for 4 h. Cell extracts were assayed for luciferase activity.

CONCLUSIONS AND SIGNIFICANCE

Previous studies have shown that PG synthesis was modulated by intracellular calcium concentration in various cells, although no mechanism was described.

On the basis of our results, we can suggest that elevation of intracellular calcium concentration stimulates GlcAT-I promoter activity primarily via the MEK/ERK pathway by targeting Sp1 binding site, which resulted in an increased binding of Sp1 factor on the promoter and eventually activated transcription (Fig. 3 ). Our data identify, for the first time, GlcAT-I as a target of calcium-dependent signaling pathway and evidence the critical role for Sp1 transcription factor in the induction of GlcAT-I expression. These data also propose that modulation of GlcAT-I promoter activity is a possible mechanism involved in the regulation of GAG synthesis by calcium-dependent pathway.

View larger version (22K): [in this window] [in a new window]   Figure 3. Proposed mechanism for the up-regulation of GlcAT-I expression by elevation of intracellular calcium and consequences on GAG chain synthesis. The rise in intracellular calcium leads to the activation of MEK/ERK pathway, which in turn stimulates GlcAT-I gene transcription by promoting the binding of Sp1 factor to the promoter. Up-regulation of GlcAT-I expression may represent a possible mechanism leading to the stimulation of GAG synthesis by calcium-dependent pathway.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/ 10.1096/fj.05-5073fje

This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.05-5073fjev1 20/10/1692    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Barré, L. Articles by Ouzzine, M. Search for Related Content PubMed PubMed Citation Articles by Barré, L. Articles by Ouzzine, M.