Parathyroid hormone (PTH), the major systemic calcium-regulating hormone, has been linked to uremic vascular changes. Considering the possible deleterious action of PTH on vascular structures, it seemed logical to evaluate the impact of PTH on the receptor of advanced glycation end products (RAGE) and interleukin 6 (IL-6) mRNA and protein expression, taking into account that such parameters might be involved in the pathogenesis of vascular calcification, atherosclerosis, and/or arteriolosclerosis. Human umbilical vein cord endothelial cells (HUVEC) were stimulated for 24 h with 10−12–10−10 mol/l PTH. The mRNA expression of RAGE and IL-6 was established by reverse transcriptase/PCR techniques. RAGE protein levels were determined by Western blot and IL-6 secretion was measured by ELISA. The pathways by which PTH may have an effect on HUVEC functions were evaluated. PTH (10−11–10−10mol/l) significantly increased RAGE mRNA and protein expression. PTH also significantly increased IL-6 mRNA expression without changes at protein levels. The addition of protein kinase (PKC or PKA) inhibitors or nitric oxide (NO) synthase inhibitors significantly reduced the RAGE and IL-6 mRNA expression and the RAGE protein expression. PTH stimulates the mRNA expressions of RAGE and IL-6 and the protein expression of RAGE. These stimulatory effects are probably through PKC and PKA pathways and are also NO dependent. Such data may explain the possible impact of PTH on the atherosclerotic and arteriosclerotic progression.
- endothelial cells
- receptor of advanced glycation end products
hypertension, vascular calcification, and atherosclerosis as well as cardiovascular morbidity and mortality are more frequent in the presence of chronic excess of parathyroid hormone (PTH), particularly in patients with end-stage renal failure (18). PTH plays a critical role in maintaining a normal calcium-phosphorus homeostasis, mainly through its impact on bones and kidneys (2). However, PTH affects the function of other organs, tissues, and cells such as endothelial cells, through membrane PTH receptors (1, 6, 7, 9, 17).
Taking into account the impact of PTH on the endothelial nitric oxide synthase (eNOS) system (17), it is conceivable that PTH may modify other endothelial cell activities. The occurrence of PTH-related vascular disease may be due to changes in the expression of factors known to be involved in the development of vasculopathies such as receptor of advanced glycation end products (RAGE) or interleukin-6 (IL-6) (5, 25, 28).
Advanced glycation end products (AGEs) are involved in the development of atherosclerosis and in the occurrence of uremic, ageing, and diabetic vascular disease (11, 15, 27). In uremia, the blood levels of AGEs are elevated (10) and endothelial RAGE is overexpressed (4). RAGE mediates the binding of AGEs to endothelial and mononuclear phagocytes and this stimulates the cell activities (20, 21).
IL-6 is considered to be one of the main mediators of inflammation as reflected by an enhanced production of fibrinogen and C-reactive protein in the liver and strongly affects the inflammatory process involved in the development of atherosclerosis through the stimulation of acute phase protein synthesis (3, 5, 25). On the basis of these data, we evaluated the possible action of PTH on gene and protein expression of RAGE and IL-6.
MATERIALS AND METHODS
Endothelial cell culture and incubation.
Endothelial cell cultures were obtained from umbilical cords as previously described (16). Ethics Review Committee approved the study and the parturient gave written informed consent. Only umbilical cords from women who had a normal pregnancy and birth were used. Cultured cells were identified as endothelial by their morphology and the presence of von Willebrand factor. Confluent cultures of human umbilical vein cord endothelial cells (HUVEC) used for experiments at passages 2–4 were incubated with different concentrations of PTH (fragment 1-34, 10−12–10−10 mol/l, equivalent to 4.1–410 pg/ml, respectively, Sigma) for 24–72 h. Each experiment included all the controls and experiment groups that were investigated.
The pathways by which PTH may have an effect on HUVEC functions were evaluated on cells pretreated for 30 min with protein kinase C (PKC) inhibitor (calphostin C, 50 nmol/l, Sigma) and/or cAMP antagonist (Rp-cAMP, 10 μmol/l, Sigma). Calphostin C inhibits PKC activity by binding to the regulatory domain of PKC (8). Rp-cAMP is a diasteromer of cAMP that competitively binds to the regulatory subunit of PKA to prevent cAMP-induced dissociation and activation of the enzyme (19). A possible involvement of nitric oxide (NO) in the PTH-induced gene expression of HUVEC was evaluated by a pretreatment with 200 μmol NG-nitro-l-arginine methyl ester (l-NAME; NOS inhibitor).
Expression of the RAGE and IL-6 genes was performed by semiquantitative multiplex RT-PCR and real-time PCR techniques. Total RNA was extracted from endothelial cells using the PUREscript RNA isolation kit (Gentra Systems), according to the manufacturer's instructions. RNA (1 μg) was then reverse transcribed into single-strand DNA with 200 U of SUPERSCRIPT II RNase Reverse Transcriptase (Invitrogen) and oligo (dT)15 primer (Promega, Madison, WI) at 37°C for 45 min, 42°C for 15 min, and 99°C for 5 min.
Semiquantitative multiplex RT-PCR amplification was performed on 1/10th of the cDNA solution with 0.5 U of Taq DNA polymerase (Sigma) at a final volume of 50 μl. The PCR conditions and primers sequence were as follows: for RAGE mRNA amplification: forward primer: 5′-CACCTTCTCCTGTAGCTTCA-3′, reverse primer: 5′-TGCCACAAGATGACCCCAAT-3′, generating a 480-bp PCR product. For IL-6 mRNA amplification: forward primer: 5′-GGTACATCCTCGACGGCATCTC-3′, reverse primer: 5′-GTTGGGTCAGGGGTGGTTATTG-3′, generating a 334-bp PCR product. β-Actin primers sequence (for semiquantitative RT-PCR of RAGE): forward primer: 5′-GAGACCTTCAACACCCCAGC-3′, reverse primer: 5′-GCTCATTGCCAATGGTGATG-3′, generating a 388-bp PCR product. β-Actin primers sequence (for semiquantitative RT-PCR of IL-6): forward primer: 5′-GACCACACCTTCTACAATGAG-3′, reverse primer: 5′-GCATACCCCTCGTAGATGGG-3′, generating a 274-bp PCR product. PCR program for IL-6: 30 cycles of 94°C for 30 s, 58°C for 40 s, and 72°C for 30 s. PCR program for RAGE: 30 cycles of 94°C for 30 s, 61°C for 30 s, and 72°C for 30 s. All primers were chosen to be complementary to domains in different exons to avoid false-positives caused by DNA contamination of the RNA preparations. RT PCR products were separated on 1.5% agarose (Sigma).
To quantify the amounts of RAGE and IL-6 mRNA expression in endothelial cells, real-time RT-PCR was performed with a Light Cycler instrument (Roche Diagnostics GmbH, Mannheim, Germany) in glass capillary tubes. The Light Cycler Fast Start DNA Master SYBR Green I reaction mix (Roche Diagnostics GmbH) and primers were added to cDNA dilutions. Primers for human IL-6 and β-actin were the same as conventional PCR. RAGE primers were: forward primer: 5′-TGGAACCGTAACCCTGACCT-3′, reverse primer: 5′-CGATGATGCTGATGCTGACA-3′. The thermal profile for SYBER Green PCRs was 95°C for 10 min, followed by 35 cycles of 95°C for 10 s, 58°C for 7 s, 72°C for 18 s, and 90°C for 5 s. To prove the specificity of the PCR product, a melting curve analysis was performed by 95°C for 5 s, 70°C for 20 s. A dilution series of a standard sample was run with the unknown samples. Gene expression was determined by normalization against β-actin expression.
Western blot analysis.
Total protein (50 μg) was subjected to electrophoresis on 7.5% SDS-polyacrylamide gels and transferred to a nitrocellulose membrane. The membrane was blocked with 5% skim milk and incubated with mouse anti-RAGE monoclonal antibody (1:1,000; Chemicon International, Temecula, CA). The second antibody was sheep anti-mouse Ig conjugated with horseradish peroxidase (Jackson ImmunoResearch Labs). The bound antibodies were visualized with enhanced chemiluminescent reporter system (ECL). The nitrocellulose membranes were stripped and blocked before being reprobed with α-tubulin monoclonal antibody (1:8,000; Sigma). The expression of RAGE was detected as a single band at 48 kDa and α-tubulin as loading control was detected as a single band at 50 kDa.
The results are expressed as means ± SE. Two-tailed Student's paired t-test was used for data analysis. P values of <0.05 were considered significant.
PTH and RAGE mRNA expression.
PTH (10−12–10−10 mol/l) significantly increased the RAGE mRNA expression after 24-h incubation (Fig. 1 and Table 1). HUVEC, pretreated with calphostin C (50 nmol/l) and/or Rp-cAMP (10 μmol/l) before PTH stimulation, significantly reduced the expression of RAGE mRNA expression [calphostin C: 30.7 ± 7.8%, P = 0.0001; Rp-cAMP: 45 ± 11.1%, P = 0.003 vs. control (PTH)] (Fig. 2). The combined inhibition with calphostin C and Rp-cAMP did not further modify the RAGE mRNA expression (36 ± 10.6%). Interestingly, the PTH-induced RAGE mRNA expression in the cells pretreated with 200 μmol l-NAME was inhibited [l-NAME: 46 ± 14.4% vs. control (PTH), P = 0.03; Fig. 2]. This interaction between NO and RAGE presently confirmed in endothelial cells has not been previously recorded in the literature.
PTH and RAGE protein expression.
We examined whether the increase in RAGE mRNA expression is associated with an increase in RAGE protein levels. PTH (10−10 mol/l) increased RAGE protein levels after 72-h incubation (Fig. 3).
PTH and IL-6 mRNA expression.
IL-6 mRNA expression was significantly increased by PTH after 24-h incubation (Fig. 5 and Table 1). Calphostin C (50 nmol/l) and/or Rp-cAMP (10 μmol/l) significantly reduced the IL-6 mRNA expression of PTH-stimulated HUVEC [calphostin C: 62.3 ± 17.7%, P = 0.01; Rp-cAMP: 70.7 ± 10%, P = 0.029 vs. control (PTH); Fig. 6]. The combined treatment of calphostin C and Rp-cAMP further reduced the IL-6 mRNA expression to 49 ± 4.8% of control (P = 0.004) but was not significant vs. calphostin C or Rp-cAMP alone. A possible involvement of NO in the PTH-induced IL-6 expression of HUVEC was evaluated by a pretreatment with 200 μmol l-NAME. Once again, l-NAME was found to inhibit IL-6 mRNA expression [l-NAME: 61.6 ± 12.8% vs. control (PTH), P = 0.039; Fig. 6]. We also examined the effect of PTH on IL-6 secretion by HUVEC. PTH had no significant effect on IL-6 secretion (10−11 mol/l: 428 ± 147 pg/ml, 10−10 mol/l: 445 ± 160 pg/ml vs. control: 472 ± 169 pg/ml, NS; results are not shown as a graph).
The present data demonstrate that PTH affects the endothelial gene expression of RAGE and IL-6 and protein levels of RAGE and that both PKA and PKC pathways are involved.
The development of vascular atherosclerosis, arteriosclerosis, and/or calcification in the presence of elevated PTH has been related partially to an increased production (and reorganization) of collagen by VSMC (12). We found that PTH could stimulate the mRNA expressions of RAGE and IL-6 and the protein expression of RAGE. PTH did not affect IL-6 secretion in HUVEC. Its impact on RAGE, which is upregulated in diabetes and uremia and is associated with higher risk of vasculopathy and atherosclerosis (21, 26, 28) and on IL-6, which is one of the main inflammatory mediators involved in the atherosclerotic disease (25), fits well with the concept that PTH may be considered an active actor in accelerating vascular atherosclerotic processes. Incubation with PKC or PKA inhibitors in the presence of PTH was associated with a reduction of RAGE mRNA and protein expression and IL-6 mRNA expression to a level equivalent to that found in nontreated cells showing that both PKC and PKA pathways may be implicated in this expression. The possible role of NO in the stimulation of RAGE and IL-6 expression induced by PTH was also estimated. Pretreatment with l-NAME inhibited RAGE and IL-6 mRNA expression and RAGE protein levels, suggesting that the elevation of RAGE and IL-6 mRNA expression could be NO dependent. As we recently demonstrated that PTH activates the eNOS system (17), it is conceivable that the parallel effects of PTH on the eNOS and RAGE and IL-6 may be interrelated.
It is well known that PTH may activate either adenylate cyclase, and subsequently PKA, or phospholipase C/PKC pathways (14, 24). In classical target cells (chondrocytes, osteoblasts, osteoclasts, and kidney-derived cells), PTH activates both pathways. In smooth muscle cells, PTH activates only the adenylate cyclase pathway (22) and may exert its vasorelaxant action via cAMP-dependent inhibition of VSMC L-type Ca2+ channel (13, 23). Our results could demonstrate that both pathways seem to be involved in PTH-related endothelial cell activation. In addition, PTH may be considered as a relevant actor in vascular remodeling processes by its stimulating action on NO release, which affects RAGE or IL-6 endothelial expression.
This work was supported by a Margaret Shtultz Grant from Tel-Aviv University.
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