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Downregulation of HD-PTP by high magnesium concentration: novel insights into magnesium-induced endothelial migration

Magnesium Research. Volume 23, Numéro 3, 119-25, september 2010, Original article

DOI : 10.1684/mrh.2010.0211


Auteur(s) : Marzia Leidi, Erika Baldoli, Jeanette AM Maier , Dipartimento di Scienze Cliniche Luigi Sacco, Università di Milano, Milano, Italy.



Auteur(s) : Marzia Leidi, Erika Baldoli, Jeanette AM Maier

Dipartimento di Scienze Cliniche Luigi Sacco, Università di Milano, Milano, Italy

Endothelial migration is an early step in angiogenesis, which is crucial in responding to tissue demands in physiological and pathological conditions [1]. Migration is also important after injuries to the vessel wall, i.e. after mechanical stress such as shear stress or cateterization, as an attempt to heal the damage [2].

Endothelial motility is triggered by growth factor receptors and integrins which activate several signalling pathways that converge on cytoskeletal remodelling to enable the progression of the migrating cells [1]. Transduction of signals by tyrosine phosphorylation is relevant in driving cell migration and results from a complex interplay between protein tyrosine kinases (PTK) and phosphatases (PTP) [1]. Among others, Focal Adhesion Kinase (FAK), a 125 kDa tyrosine kinase, and Src, a non receptor tyrosine kinase, are crucial in the regulation of cell motility because they modulate the turnover of focal adhesions [3]. PTPs contribute to the regulation of endothelial migration, as underscored by the results showing that PTP inhibitors promote neovascularization in vitro and in vivo [4]. The expression of some PTPs have been demonstrated in endothelial cells. In particular, PTPη causes vascular defects when mutated, while PTPε promotes endothelial migration by activating c-Src [5]. In endothelial cells derived from different vascular beds we have demonstrated the expression of HD-PTP (PTPN23), a non-transmembrane protein tyrosine phosphatase which possesses a classical tyrosine phosphatase domain [6]. However, controversy exists about its catalytic activity [7, 8], and until now no HD-PTP phosphorylated substrate has been identified. We have demonstrated that the angiogenic molecule basic Fibroblast Growth Factor (FGF), which induces endothelial migration, downregulates HD-PTP via proteasome [9]. Accordingly, endothelial cells silencing HD-PTP after transfection with small interference RNA migrate more than controls [10]. We therefore argue for a role of HD-PTP in inhibiting endothelial migration.

Since magnesium (Mg) is a chemoattractant for endothelial cells [11, 12], we evaluated whether high concentrations of Mg affect the levels of HD-PTP, FAK and Src. In this report, we show that the downregulation of HD-PTP correlates with the induction of endothelial motility upon culture in high concentrations of Mg.

Materials and methods

Cell culture and migration

Human umbilical vein endothelial cells (HUVEC) were isolated by collagenase digestion and cultured in M199 containing 20% fetal bovine serum, 1 mM glutamine, 1 mM penicillin and streptomycin, Endothelial Cell Growth Factor (150 μg/mL), 1 mM sodium pyruvate and heparin (5 units/mL) on 2% gelatin-coated dishes. The cells were routinely evaluated for the expression of endothelial markers, i.e. endothelial nitric oxide synthase and CD34, and utilized for 5-6 passages. To increase the concentration of Mg in the culture media, MgSO4 was used. No significant difference was observed whether we used MgSO4 or MgCl2 to increase Mg concentrations in our culture media.

Confluent HUVEC were cultured in the presence of different concentrations of Mg for 2 days. Migration was determined using an in vitro model of wound repair as previously described [7]. After wounding, the monolayer was washed and incubated for 24 additional h in the corresponding concentration of Mg, before staining with crystal violet. The wound area was calculated by the ImageJ software and expressed using an arbitrary value scale. The experiments were performed in triplicate. Data are shown as the mean ± standard deviation (SD).

Reverse transcription (RT)-PCR

Cells were lysed in 4 M guanidinium isothiocyanate and RNA was purified as described [9]. For RT-PCR, 1 μg of RNA was reverse transcribed and PCR amplification was carried out using 1/50 of the final RT reaction. Each amplification cycle consisted of 30 sec at 95°C, 30 sec at 52°C and 1 min at 72°C using 30 pmol of each primer. The reaction was stopped after 30 cycles. One fifth of the reaction mix was separated on a 1% agarose gel. The sequences of the HD-PTP primers are the following: sense 5’-GCTGCAGCAGCTACGGGAGTGG-3’ and antisense 5’-CTCCTTTTACAGGCTGAAGAGTGTC-3’ [9]. RT-PCR with specific primers for actin was performed to normalize (sense 5’-GCATGGAGTCCTGTCGCATCC-3’ antisense 5’-GCGGCCAGGATGGAGCCGC-3’).

Western blot analysis

Cell extracts (100 μg/lane) were resolved on 8% SDS-polyacrylamide gels, transferred to nitrocellulose sheets at 250 mA for 16 h, and probed with anti-HD-PTP IgGs (10 μg/mL) [7]. Antibodies against the phosphorylated tyrosine 419 (pY419) of Src were from Cell Signaling Technology (Boston, MA). Antibodies against the phosphorylated tyrosine 397 (pY397) of FAK were from Upstate (Prodotti Gianni, Milano, Italy). Antibodies against Src, actin and FAK were from Santa Cruz (Tebu-bio, Magenta, Italy). Secondary antibodies were labelled with horseradish peroxidase (Pierce, Rockford IL, USA). The SuperSignal chemiluminescence kit (Pierce) was used to detect immunoreactive proteins. All the Western blots were repeated at least three times.

Statistical analysis

All experiments were repeated at least three times in triplicate. Data are presented as means ± SD. Statistical differences were determined using the unpaired two-tailed Student's t test (p < 0.001).


High concentrations of Mg stimulate HUVEC migration

Confluent HUVEC were cultured in different concentrations for 48 h. The cell monolayer was then wounded and migration assessed after 24 h [10]. Figure 1A shows a typical wound assay performed on HUVEC in physiological (1.0 mM) or in high (5.0 mM) concentrations of extracellular Mg, immediately after the wound (0h) and 24h later (24h). HUVEC migration was assessed in the presence of 1.0, 2.0, 3.0 and 5.0 mM Mg as described above. Figure 1B shows that Mg stimulation of endothelial migration was dose dependent.

High concentrations of Mg do not modulate FAK and Src total amounts and phosphorylation

In HUVEC cultured in Mg-deficient medium, we have shown the downregulation of Src [13], which is required for endothelial migration. Here we demonstrate that culture in high Mg containing medium for 72h did not determine significant alterations of Src total amounts (figure 2). Another molecule implicated in focal adhesion turn-over and, therefore, in migration is FAK. We did not detect any modulation of the total amounts of FAK in HUVEC cultured in 5.0 mM Mg for 72h (figure 3).

Src, which is activated upon the phosphorylation of its Tyr419 [14], phosphorylates FAK on Tyr397 and activates it. We therefore investigated the extent of Src and FAK tyrosine phosphorylation in HUVEC in 5.0 mM Mg vs controls in 1.0 mM Mg. We found no modulation of Tyr419-Src and Tyr397-FAK by high extracellular Mg as detected by western blot (figures 2 and 3).

High concentrations of Mg downregulate HD-PTP

We have previously shown a role of HD-PTP in modulating endothelial migration. In particular, endothelial cells silencing HD-PTP migrate faster than controls [10]. We therefore evaluated whether high extracellular Mg altered the total amounts of HD-PTP. We cultured HUVEC in 5.0 mM for different times. By RT-PCR we found no modulation of HD-PTP transcript (figure 4A), whereas by western blot we observed a marked downregulation of HD-PTP after 16 h of culture in 5.0 mM Mg, which is maintained up to 48 h (figure 4B).

We described the downregulation of HD-PTP by FGF through degradation via the proteasome [9]. We therefore exposed HUVEC cultured in 5.0 mM Mg to the proteasome inhibitor MG132, which has been widely used to turn off the proteasome [9]. HD-PTP remained unchanged after treatment with MG132 (5 μM) (figure 4C).


High concentrations of extracellular Mg stimulate endothelial migration [11, 12]. Several mechanisms could be involved. Mg upregulates integrin function and is also required for the assembly of actin polymers and for myosin ATPase activity, two key components of the motor responsible for cell migration [15]. It has also been demonstrated that the induction of endothelial motility by high Mg levels is not due to the regulation of energy production, but it is the consequence of receptor-mediated events implicating heterotrimeric G-binding proteins, protein kinase C and tyrosine kinase signalling [11].

Overall tyrosine phosphorylation is the result of the coordinated actions of PTK and PTP and is instrumental for the motile process [1]. We first investigated whether high extracellular Mg modulated the total amounts or the activation of two tyrosine kinases, namely Src and FAK. Src is a key factor in the regulation of cell motility because, after activation by phosphorylation of its Tyr419 [14], it activates FAK by phosphorylating Tyr397. Activated FAK and Src then cooperate in focal adhesion turnover which is a pre-requisite for cell migration. We found no modulation of either the total amounts or the degree of phosphorylation of FAK and Src in HUVEC cultured in high Mg containing medium.

PTPs are thought to play a role in regulating endothelial migration [4, 5]. Recently, we have proposed that HD-PTP participates in the control of endothelial motility [7, 10]. Indeed, endothelial cells silencing HD-PTP acquire a scattered and spindle-shaped phenotype and migrate more than controls. However, how HD-PTP participates in the regulation of cell motility has not been fully elucidated. Based on its amino acid sequence, HD-PTP has been classified as a non transmembrane PTP, but at the moment no HD-PTP phosphorylated substrate has been identified. Apart from its still controversial enzymatic activity, we hypothesize that HD-PTP might control cell signaling pathways by interacting with different proteins. Interestingly, HD-PTP interacts with Src and FAK [7, 10]. In this paper we show that the induction of HUVEC migration correlates with the downregulation of HD-PTP, thus mimicking the silencing obtained with the siRNA [7, 10]. In HUVEC cultured in high Mg, we hypothesize that Src and FAK do not interact with HD-PTP and become available to be activated and, consequently, to induce the acquisition of a migratory phenotype.

The decrease in the total amounts of HD-PTP by high Mg concentration cannot be ascribed to transcriptional regulation since the HD-PTP transcript is comparable in HUVEC cultured in 1.0 and in 5.0 mM Mg. It is noteworthy that the angiogenic molecule FGF, which is chemotactic for HUVEC [1], downregulates HD-PTP through the proteasome [9]. However, in HUVEC cultured in high Mg containing medium we rule out a role of the proteasome, because MG132 did not restore the levels of HD-PTP. This result indicates that other proteases are involved in the regulation of HD-PTP. Further studies are necessary to elucidate the mechanisms by which high Mg concentration reduces the total amounts of HD-PTP.

A puzzling question that remains to be answered is the contribution of transient receptor potential melastatin-like (TRPM)-7 to the modulation of endothelial migration. Under physiological conditions, TRPM-7, which encodes a divalent cation channel fused to an alpha-kinase domain, preferentially transports Mg [16]. TRPM-7 kinase activity has been shown to remodel the actomyosin cytoskeleton thus leading to podosome formation in neuroblastoma cells [17]. Accordingly, silencing TRPM-7 decreases the migratory ability of nasopharyngeal carcinoma cells [18]. On these bases, it is tempting to speculate about a possible link between TRPM-7 and endothelial migration. Further investigation into whether and how TRPM7 regulates endothelial migration will be important to pursue.Financial support and disclosure

This work is supported by MIUR-PRIN 2007 grant number 2007ZT39FN.

None of the authors has any conflict of interest to disclose.


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