1α,25(OH)2D3 regulates pro-angiogenic factors in endothelial cells transformed by Kaposi's sarcoma-associated herpesvirus G protein coupled receptor.

When tumoral cell expansion exceeds the vascular supply, regions of hypoxia or low oxygen concentration are generated promoting the formation of new vessels through cell proliferation and migration. Viral G protein-coupled receptor (vGPCR) is associated to Kaposi's sarcoma pathology and induces a paracrine transformation when is stably expressed in murine endothelial cells activating hypoxia-induced transcription factors. Previously, we reported the antiproliferative actions of 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) in endothelial cells transformed by the vGPCR (SVEC-vGPCR). Herein, we further investigated if pro-angiogenic factors as AP-1, HIF-1α and VEGF are modulated by 1α,25(OH)2D3. We found by qRT-PCR analysis that the mRNA level of JunB, a negative regulator of cell proliferation, was similarly increased at all-time points tested after 1α,25(OH)2D3 treatment in SVEC-vGPCR cells. Also, mRNA levels of the pro-angiogenic factor c-Fos, which induces tumor invasion, were only decreased during one short period treatment. In addition, Hif-1α mRNA and protein levels were significantly reduced after 1α,25(OH)2D3 treatment in a VDR dependent fashion. However, mRNA levels of the angiogenic activator Vegf, promoted in turn by Hif-1α expression, were surprisingly high depending on VDR expression as well. Moreover, Egr-1, which has been reported to induce VEGF expression independently of HIF-1α, diminished its expression with 1α,25(OH)2D3 treatment, fact that was related to the decline of p-ERK1/2. Altogether, these results suggest a negative modulation of some pro-angiogenic factors like AP-1 and HIF-1α, as part of the antiproliferative mechanism of 1α,25(OH)2D3 in SVEC-vGPCR endothelial cells.

The Stability Landscape of de novo TIM Barrels Explored by a Modular Design Approach.

The ability to design stable proteins with custom-made functions is a major goal in biochemistry with practical relevance for our environment and society. Understanding and manipulating protein stability provide crucial information on the molecular determinants that modulate structure and stability, and expand the applications of de novo proteins. Since the (ß/⍺)8-barrel or TIM-barrel fold is one of the most common functional scaffolds, in this work we designed a collection of stable de novo TIM barrels (DeNovoTIMs), using a computational fixed-backbone and modular approach based on improved hydrophobic packing of sTIM11, the first validated de novo TIM barrel, and subjected them to a thorough folding analysis. DeNovoTIMs navigate a region of the stability landscape previously uncharted by natural TIM barrels, with variations spanning 60 degrees in melting temperature and 22 kcal per mol in conformational stability throughout the designs. Significant non-additive or epistatic effects were observed when stabilizing mutations from different regions of the barrel were combined. The molecular basis of epistasis in DeNovoTIMs appears to be related to the extension of the hydrophobic cores. This study is an important step towards the fine-tuned modulation of protein stability by design.

Down-regulation of COX-2 activity by 1α,25(OH)2D3 is VDR dependent in endothelial cells transformed by Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor.

Our previous reports showed that 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) has antiproliferative actions in endothelial cells stably expressing viral G protein-coupled receptor (vGPCR) associated with the pathogenesis of Kaposi's sarcoma. It has been reported that COX-2 enzyme, involved in the tumorigenesis of many types of cancers, is induced by vGPCR. Therefore, we investigated whether COX-2 down-regulation is part of the growth inhibitory effects of 1α,25(OH)2D3. Proliferation was measured in presence of COX-2 inhibitor Celecoxib (10-20 µM) revealing a decreased in vGPCR cell number, displaying typically apoptotic features in a dose dependent manner similarly to 1α,25(OH)2D3. In addition, the reduced cell viability observed with 20 µM Celecoxib was enhanced in presence of 1α,25(OH)2D3. Remarkably, although COX-2 mRNA and protein levels were up-regulated after 1α,25(OH)2D3 treatment, COX-2 enzymatic activity was reduced in a VDR-dependent manner. Furthermore, an interaction between COX-2 and VDR was revealed through GST pull-down and computational analysis. Additionally, high-affinity prostanoid receptors (EP3 and EP4) were found down-regulated by 1α,25(OH)2D3. Altogether, these results suggest a down-regulation of COX-2 activity and of prostanoid receptors as part of the antineoplastic mechanism of 1α,25(OH)2D3 in endothelial cells transformed by vGPCR.

In vitro studies revealed a downregulation of Wnt/ß-catenin cascade by active vitamin D and TX 527 analog in a Kaposi's sarcoma cellular model.

The Kaposi's sarcoma-associated herpesvirus G-protein-coupled receptor (vGPCR) is a key molecule in the pathogenesis of Kaposi's sarcoma. We have previously demonstrated that 1α,25(OH)2D3 or its less calcemic analog TX 527 exerts antiproliferative effects in endothelial cells stable expressing vGPCR. Since it is well documented that vGPCR activates the canonical Wnt/ß-catenin signaling pathway, the aim of this study was to evaluate if Wnt/ß-catenin cascade is target of 1α,25(OH)2D3 or TX 527 as part of their antineoplastic mechanism. Firstly, Western blot studies showed an increase in ß-catenin protein levels in a dose and time dependent manner; and when VDR was knockdown, ß-catenin protein levels were significantly decreased. Secondly, ß-catenin localization, investigated by immunofluorescence and subcellular fractionation techniques, was found increased in the nucleus and plasma membrane after 1α,25(OH)2D3 treatment. VE-cadherin protein levels were also increased in the plasma membrane fraction. Furthermore, ß-catenin interaction with VDR was observed by co-immunoprecipitation and mRNA expression of ß-catenin target genes was found decreased. Finally, DKK-1, the extracellular inhibitor of Wnt/ß-catenin pathway, showed an initial upregulation of mRNA expression. Altogether, the results obtained by different techniques revealed a downregulation of Wnt/ß-catenin cascade after 1α,25(OH)2D3 or TX 527 treatment, showing the foundation for a potential chemotherapeutic agent.

VDR agonists down regulate PI3K/Akt/mTOR axis and trigger autophagy in Kaposi's sarcoma cells.

The Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor (KSHV/vGPCR) is a key molecule in the pathogenesis of Kaposi's sarcoma. We have previously shown that 1α,25(OH)2D3 or its less-calcemic analog TX 527 inhibits the proliferation of endothelial cells expressing vGPCR, NF-κB activity and induces apoptosis in a VDR dependent manner. In this work, we further explored whether 1α,25(OH)2D3 or TX 527 regulates PI3K/Akt/mTOR axis and induces autophagy as part of its antineoplastic mechanism of action. Proliferation assays indicated that vGPCR cell number decreased in presence of LY294002 (PI3K/Akt inhibitor) likewise 1α,25(OH)2D3 or TX 527 (10 nM, 48 h). Also, Akt phosphorylation was found decreased in dose (0.1-100 nM) and time response studies (12-72 h) after both compounds treatments. In addition, decreased phosphorylated Akt was significantly observed in the nucleus. Moreover, regulation of Akt phosphorylation was NF-κB and VDR dependent. TNFAIP3/A20, an ubiquitin-editing enzyme, a direct NF-κB target gene and a negative regulator of Beclin-1, was down-regulated whereas Beclin-1 was up-regulated after 10 nM of 1α,25(OH)2D3 or TX 527 treatment. Decrement in Akt phosphorylation was accompanied by a reduced mTOR phosphorylation and an increase in the autophagy marker LC3-II. Since increment in autophagosomes not always indicates increment in autophagy activity, we used Chloroquine (CQ, 1 µM), an inhibitor of autophagy flow, to confirm autophagy after both VDR agonists treatment. In conclusion, VDR agonists, 1α,25(OH)2D3 or TX 527, inhibited PI3K/Akt/mTOR axis and induced autophagy in endothelial cells expressing vGPCR by a VDR-dependent mechanism.

Antineoplastic effect of 1α,25(OH)2D3 in spheroids from endothelial cells transformed by Kaposi's sarcoma-associated herpesvirus G protein coupled receptor.

The Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor (KSHV/vGPCR) is a key molecule in the pathogenesis of Kaposi's sarcoma. In endothelial cells, tumor maintenance and NF-κB activation depends on vGPCR constitutive expression and activity. We have previously demonstrated that 1α,25(OH)2D3 induces apoptosis in a VDR dependent manner, inhibits vGPCR cell growth and NF-κB activity. In this study, we developed a method to obtain multicellular spheroids (MCS) from endothelial cells expressing vGPCR in order to test whether MCS have a similar response to 2D-cultures after 1α,25(OH)2D3 treatment. Firstly, we found that vGPCR MCS started to form at 2nd day-growth, reaching a diameter up to 300 µm at 7th day-growth, whereas cells without vGPCR expression (SVEC) developed spheroids earlier and remained smaller throughout the period monitored. Secondly, vGPCR MCS size and architecture were analyzed during 1α,25(OH)2D3 (0.1-100 nM, 48 h) treatment. We found that once treated with 10 nM of 1α,25(OH)2D3 the initials MCS began a slight disaggregation with no changes in size; whereas at the higher dose (100 nM) the architecture of MCS was found completely broken. Furthermore, VDR mRNA expression increased significantly and this change was accompanied by a reduction of HIF-1α, an increase of VEGF, p21 and Bim mRNA expression. Finally, results from Western blot analysis showed that 1α,25(OH)2D3 decreased Akt and ERK1/2 protein phosphorylation. In conclusion, these data have revealed that 1α,25(OH)2D3 inhibits vGPCR MCS proliferation and induces apoptosis similar to vGPCR cells growing in 2D-cultures.