Study of the regulation of the expression and activities of heparan 3-O-sulfotransferases 2, 3A and 3B in macrophages

Les héparanes sulfates (HS) sont des polysaccharides sulfatés qui participent à de nombreux processus physiopathologiques. L’entrée du virus HSV-1 dans ses cellules cibles nécessite l’intervention de la glycoprotéine gD, qui peut interagir avec quatre récepteurs différents: HVEM (Herpes Virus Entry Mediator), les nectines-1 et -2, et des HS 3-O-sulfatés. De plus, la protéine gD est impliquée dans la protection des cellules cibles contre l’apoptose. Nos travaux ont montré que les réponses anti-apoptotiques induites par la gD nécessitent la coopération entre HVEM et les HS 3-O-sulfatés. Chez l’Homme, sept 3-O-sulfotransférases (3-OSTs) sont impliquées dans la réaction de 3-O-sulfatation, qui se distinguent par des différences d’expression et de spécificité de substrat. Selon l'environnement inflammatoire, les macrophages subissent deux types de polarisation, caractérisés par des phénotypes et des fonctions distinctes. Les travaux du Laboratoire ont montré que la 3-OST3B est fortement induite dans les macrophages pro-inflammatoires, à l’inverse de la 3-OST2. Nous avons alors montré que l’expression de la 3-OST3B est régulée de manière transcriptionnelle et post-transcriptionnelle. En revanche, la diminution de la 3-OST2 fait intervenir des mécanismes différents, dont l’action d’un microARN. La dernière partie de la thèse a porté sur la localisation subcellulaire des 3-OSTs. Nos résultats montrent que la 3-OST3B est localisée dans le Golgi, alors que la 3-OST2 et la 3-OST3A sont retrouvées jusqu’à la membrane plasmique. Dans leur ensemble, nos résultats suggèrent une fonction spécifique pour chaque 3-OST dans la génération de motifs HS 3-O-sulfatés distincts.Heparan sulfates (HS) are sulfated polysaccharides able to modulate several normal and pathological processes. Binding of Herpes-Simplex virus HSV-1 to its target cells is mediated in part by the glycoprotein gD. This protein can interact with four receptors: HVEM (Herpes Virus Entry Mediator), nectin-1 and -2, and 3-O-sulfated HS. In addition, gD is capable of protecting host cells against apoptosis. In this context, we showed that gD protects macrophages against apoptosis by a mechanism dependent on the cooperation between HVEM and 3-O-sulfated HS. The reaction of 3-O-sulfation can be catalyzed by seven 3-O-sulfotransferases (3-OSTs), which are differentially expressed and exhibit fine substrate specificity. Depending on the immune environment, macrophages can undergo proinflammatory (M1) or alternative (M2) polarization, resulting in distinct phenotypes and functions. A previous study of the laboratory has showed that 3-OST3B is strongly induced in M1 macrophages, while 3-OST2 is poorly detected. We then demonstrated that the induction of 3-OST3B is regulated at the transcriptional and post-transcriptional levels upon M1 polarization. Conversely, the reduction of 3-OST2 expression involved distinct mechanisms, including the action of a microRNA.The last part of the thesis focused on the subcellular localization of 3-OSTs. Our results demonstrated that 3-OST3B is localized in the Golgi apparatus, while 3-OST2 and 3-OST3A reach the plasma membrane. Altogether, our results suggest that each 3-OST isoenzyme may be involved in the generation of distinct 3-O-sulfated HS motifs

Effects of the expression of HS3ST2, HS3ST3B and HS3ST4 on colony formation in MDA-MB-231 cells.

<p>(<b>A</b>) Equal numbers of control and HS3ST-overexpressing cells (2000 per well) were seeded in six well plates and maintained for nine days in DMEM complemented with 1% FCS to form colonies. Fresh complete growth medium was then added for three days, after which the colonies were stained with crystal violet. The right panel (<b>B</b>) represents the quantification of the colonies per well. Results are expressed as fold changes by comparison with control cells transfected with empty vector. Data are means ± S.D. from five separate experiments performed independently (***<i>P</i> < 0.001, significantly different when compared to control cells).</p

Overexpression of gD-type HS3STs in MDA-MB-231 cells.

<p>Cells were transiently transfected with empty vector (control) or vectors encoding HS3ST2, HS3ST3A, HS3ST3B or HS3ST4, and then cultured for 24 h and 48 h prior analysis of the expression and activity of each isozyme. (<b>A</b>) Following RNA extraction, the mRNA levels of HS3STs were quantified by real-time RT-PCR at 24 h post-transfection. Relative abundance of the transcripts was normalized to endogenous HPRT mRNA. Data are means ± SD from at least three distinct experiments. (<b>B</b>) In parallel experiments, proteins from cell lysates were separated by SDS-PAGE and subjected to Western blotting with appropriate specific antibodies. Parallel immunoblotting with antibodies to GAPDH confirmed equal loading of the samples. (<b>C</b>) Efficiency in the reaction of heparan 3-<i>O</i>-sulfation was confirmed 48 h post-transfection by using HS4C3 antibody. Cell surface fluorescent staining was detected by flow cytometry. Filled histograms represent the staining obtained with HS4C3 antibody, while open histograms correspond to the binding of the irrelevant antibody MBP49. Each panel represents the binding of the antibodies to the surface of HS3ST-expressing cells (black histograms) compared to the binding of the same antibodies to control cells (grey histograms). Data are representative of three separate experiments. (<b>D</b>) Compositional disaccharide analysis of HS from transfected MDA-MB-231 cells. Following depolymerization of HS with a mixture of heparinases, disaccharides were labelled with AMAC and resolved by RP-HPLC (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194676#pone.0194676.s001" target="_blank">S1 Fig</a>). Data are expressed as frequency of each disaccharide and correspond to means ± S.D. from two sets of triplicate injections performed with distinct cell preparations (**<i>P</i> < 0.01, ***<i>P</i> < 0.001, significantly different when compared to control cells).</p

Effects of the expression of HS3STs on NK-mediated cell death.

<p>Control or HS3ST-overexpressing MDA-MB-231 cells were cultured for 32 h and then exposed to activated NK cells for 5 h at the different ratios target/effector 1:1, 1:5 and 1:10. NK-mediated cytotoxicity was estimated by using a LDH assay, as described in “Materials and Methods”. Data are means ± S.D. from three separate experiments performed with NK cells from distinct donors (*<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001, significantly different when compared to control cells; <i>n</i>.<i>s</i>., not significantly different).</p

Effects of the expression of HS3ST2, HS3ST3A, HS3ST3B and HS3ST4 on breast cancer cell growth.

<p>MDA-MB-231 and BT-20 cells were transfected with overexpression vectors and cultured in medium containing 1% FCS for 24 h and 48 h. At each time, the effects of HS3STs on the growth of MDA-MB-231 and BT-20 cells was estimated by cell counting (<b>A</b>) and MTS assay (<b>B</b>). Results are expressed as fold changes by comparison with control cells transfected with empty vector. Data are means ± S.D. from three separate experiments performed independently (*<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001, significantly different when compared to control cells).</p

The heparan sulfate 3-<i>O</i>-sulfotransferases (HS3ST) 2, 3B and 4 enhance proliferation and survival in breast cancer MDA-MB-231 cells

<div><p>Heparan sulfate 3-<i>O</i>-sulfotransferases (HS3STs) catalyze the final maturation step of heparan sulfates. Although seven HS3ST isozymes have been described in human, 3-<i>O</i>-sulfation is a relatively rare modification, and only a few biological processes have been described to be influenced by 3-<i>O</i>-sulfated motifs. A conflicting literature has recently reported that HS3ST2, 3A, 3B and 4 may exhibit either tumor-promoting or anti-oncogenic properties, depending on the model used and cancer cell phenotype. Hence, we decided to compare the consequences of the overexpression of each of these HS3STs in the same cellular model. We demonstrated that, unlike HS3ST3A, the other three isozymes enhanced the proliferation of breast cancer MDA-MB-231 and BT-20 cells. Moreover, the colony forming capacity of MDA-MB-231 cells was markedly increased by the expression of HS3ST2, 3B and 4. No notable difference was observed between the three isozymes, meaning that the modifications catalyzed by each HS3ST had the same functional impact on cell behavior. We then demonstrated that overexpression of HS3ST2, 3B and 4 was accompanied by increased activation of c-Src, Akt and NF-κB and up-regulation of the anti-apoptotic proteins survivin and XIAP. In line with these findings, we showed that HS3ST-transfected cells are more resistant to cell death induction by pro-apoptotic stimuli or NK cells. Altogether, our findings demonstrate that HS3ST2, 3B and 4 share the same pro-tumoral activity and support the idea that these HS3STs could compensate each other for loss of their expression depending on the molecular signature of cancer cells and/or changes in the tumor environment.</p></div

Protective effects of the expression of HS3ST2, HS3ST3B and HS3ST4 against apoptosis.

<p>Transfected MDA-MB-231 cells were treated with staurosporin (1 μM) for 5 h or with a mixture of anti-Fas antibody (100 ng/mL) and TNF-α (100 ng/mL) for 16 h. (<b>A</b>) The percentage of apoptotic cell population was evaluated by analyzing the binding of fluorescein-conjugated annexin-V. Each bar of histogram represents mean ± S.D. of the rate of apoptotic cells by comparison with untreated control cells. Data were obtained from three distinct experiments. (<b>B</b>) In parallel experiments, the activation of caspase-3 was analysed in HS3ST-overexpressing cells and in control cells after exposure to either staurosporin or to the mixture of anti-Fas antibody plus TNF-α. Results are expressed as fold increase in caspase-3 activity by comparison with untreated control cells and correspond to means ± S.D. from three independent experiments (*<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001, significantly different when compared to control cells; <i>n</i>.<i>s</i>., not significantly different).</p

Effect of the expression of HS3ST2, HS3ST3B and HS3ST4 on cell signaling.

<p>Twenty hours post transfection, MDA-MB-231 cells were serum-starved for 3 hours, collected and lysed. Proteins were then separated by SDS-PAGE and subjected to Western blotting with antibodies to I-κB, survivin, XIAP and to the phosphorylated forms of c-Src, Akt, ERK1/2, STAT3 and NF-κB p65 subunit. Parallel immunoblotting with antibodies to GAPDH and to c-Src, Akt, ERK1/2, STAT3 and NF-κB p65 regardless of their phosphorylation status confirmed equal loading of samples. Histograms represent the quantification of the phosphorylation status of c-Src, Akt, ERK1/2, STAT3, NF-κB p65 and of the expression of I-κB, survivin and XIAP related to GAPDH. Data were normalized to control cells. Representative results from three independent experiments are shown.</p

Heparan sulfate 3- O -sulfotransferase 2 (HS3ST2) displays an unexpected subcellular localization in the plasma membrane

Background: Heparan sulfate (HS) 3-O-sulfation can be catalysed by seven 3-O-sulfotransferases (HS3STs) in humans, still it is the rarest modification in HS and its biological function is yet misunderstood. HS3ST2 and HS3ST3B exhibit the same activity in vitro. They are however differently expressed in macrophages depending on cell environment, which suggests that they may be involved in distinct cellular processes. Here, we hypothesized that both isozymes might also display distinct subcellular localizations.Methods: The subcellular distribution of HS3ST2 and HS3ST3B was analysed by using overexpression systems in HeLa cells. The localization of endogenous HS3ST2 was confirmed by immunostaining in primary macrophages.Results: We found that HS3ST3B was only localized in the Golgi apparatus and no difference between full-length enzyme and truncated construct depleted of its catalytic domain was observed. In contrast, HS3ST2 was clearly visualized at the plasma membrane. Its truncated form remained in the Golgi apparatus, meaning that the catalytic domain might support correct addressing of HS3ST2 to cell surface. Moreover, we found a partial co-localization of HS3ST2 with syndecan-2 in HeLa cells and primary macrophages. Silencing the expression of this proteoglycan altered the localization of HS3ST2, which suggests that syndecan-2 is required to address the isozyme outside of the Golgi apparatus.Conclusions: We demonstrated that HS3ST3B is a Golgi-resident isozyme, while HS3ST2 is addressed to the plasma membrane with syndecan-2.General significance: The membrane localization of HS3ST2 suggests that this enzyme may participate in discrete processes that occur at the cell surface

Treatment and Prognosis of Patients with Lung Cancer and Combined Interstitial Lung Disease.

Interstitial lung disease (ILD) is associated with a higher lung cancer (LC) risk and may impact cancer's clinical characteristics, treatment strategies, and outcomes. This impact's extent is unclear, particularly in Caucasians. In this retrospective observational study, we reviewed the files of all LC patients diagnosed in a 38-month period. Expert radiologists reviewed the computed tomography scans performed at diagnosis. Patients with LC and ILD ( = 29, 7%) were compared to those without ILD ( = 363, 93%) for population and cancer characteristics, treatments, and clinical outcomes. Patients with LC and ILD were older (73 ± 8 vs. 65 ± 11 years; < 0.001). There was no significant difference in LC histology, localization, stage, or treatment modalities. The respiratory complication rate after cancer treatment was significantly higher in the ILD group (39% vs. 6%; < 0.01). Overall survival rates were similar at 12 (52% vs. 59%; = 0.48) and 24 months (41% vs. 45%; = 0.64) but poorer in the ILD group at 36 months, although not statistically significant (9% vs. 39%; = 0.06). The ILD group had a higher probability of death (hazard ratio (HR) = 1.49 [0.96;2.27]), but this was not statistically significant ( = 0.06). In a Cox regression model, patients with ILD treated surgically had a significantly higher mortality risk (HR = 2.37 [1.1;5.09]; = 0.03). Patients with combined LC and ILD have worse clinical outcomes even when similar treatment modalities are offered