In the present work we have functionally characterized these ligand binding regions. Similar to the wild type receptor, both regions bind TGF-␤2 with higher affinity than TGF-␤1. However, only the endoglin-related region increases the TGF-␤2 labeling of the TGF-␤ type II receptor, the so-called "TGF-␤ -presentation" function of the wild type receptor. Despite this preference, both regions as well as the wild type receptor mediate the TGF-␤2-dependent Smad2 phosphorylation, indicating that they can function indistinguishably as TGF-␤-enhancing coreceptors. On the other hand, we found that the recently described ability of the wild type betaglycan to bind inhibin A is a property of the core protein that resides in the uromodulin-related region. Binding competition experiments indicate that this region binds inhibin and TGF-␤ with the following relative affinities: TGF-␤2 > inhibin A > TGF-␤1. All together, the present results suggest that betaglycan ectodomain is endowed with two bona fide independent ligand binding domains that can perform specialized functions as co-receptors of distinct members of the TGF-␤ superfamily. Transforming growth factor-␤ (TGF-␤)1 is the prototype of a superfamily of growth factors involved in the regulation of cell proliferation, differentiation, and development (1, 2). TGF-␤ signals through a complex of transmembrane serine/threonine kinase receptors, the TGF-␤ type I and type II receptors. Ligand binding promotes the association between the type I and II receptors. In this complex, phosphorylation of the type I receptor kinase by the constitutively active type II receptor kinase results in its activation. Active type I receptor phosphorylates members of a novel family of transcriptional regulators, the Smads, which transduce the TGF-␤ signal into the cell nucleus (3, 4).TGF-␤ has two known co-receptors, betaglycan and endoglin, which are transmembrane glycoproteins with large extracellular regions that bind TGF-␤ and small cytoplasmatic regions without any clearly identifiable signaling motif (5-8). Betaglycan is a membrane proteoglycan containing heparan and chondroitin sulfate chains whose core protein binds all three TGF-␤ isoforms (9 -11). Betaglycan is capable of fine tuning the availability of TGF-␤ to the signaling receptors, thereby determining the outcome of the TGF-␤ stimulation (12, 13). This regulation is both positive and negative. Although the membrane-bound form of betaglycan increases the binding of TGF-␤ to the signaling complex, the soluble form of betaglycan prevents this binding and therefore blocks the actions of TGF-␤ (14). These effects are more dramatic for TGF-␤2, the isoform for which betaglycan has higher affinity (15-17). Expression of membrane betaglycan in cells that normally do not express this co-receptor increases their binding to TGF-␤2 and corrects for their low sensitivity to this TGF-␤ isoform (18,19). Presumably, this effect is mediated by a TGF-␤-induced "presentation complex" formed between membrane-bound betaglycan and the TGF-␤ type II rece...

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BackgroundApproximately 30% of breast tumors do not express the estrogen receptor (ER) α, which is necessary for endocrine therapy approaches. Studies are ongoing in order to restore ERα expression in ERα-negative breast cancer. The aim of the present study was to determine if calcitriol induces ERα expression in ER-negative breast cancer cells, thus restoring antiestrogen responses.MethodsCultured cells derived from ERα-negative breast tumors and an ERα-negative breast cancer cell line (SUM-229PE) were treated with calcitriol and ERα expression was assessed by real time PCR and western blots. The ERα functionality was evaluated by prolactin gene expression analysis. In addition, the effects of antiestrogens were assessed by growth assay using the XTT method. Gene expression of cyclin D1 (CCND1), and Ether-à-go-go 1 (EAG1) was also evaluated in cells treated with calcitriol alone or in combination with estradiol or ICI-182,780. Statistical analyses were determined by one-way ANOVA.ResultsCalcitriol was able to induce the expression of a functional ERα in ER-negative breast cancer cells. This effect was mediated through the vitamin D receptor (VDR), since it was abrogated by a VDR antagonist. Interestingly, the calcitriol-induced ERα restored the response to antiestrogens by inhibiting cell proliferation. In addition, calcitriol-treated cells in the presence of ICI-182,780 resulted in a significant reduction of two important cell proliferation regulators CCND1 and EAG1.ConclusionsCalcitriol induced the expression of ERα and restored the response to antiestrogens in ERα-negative breast cancer cells. The combined treatment with calcitriol and antiestrogens could represent a new therapeutic strategy in ERα-negative breast cancer patients.

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BackgroundThe oncogenic ether-à-go-go-1 potassium channel (EAG1) activity and expression are necessary for cell cycle progression and tumorigenesis. The active vitamin D metabolite, calcitriol, and astemizole, a promising antineoplastic drug, target EAG1 by inhibiting its expression and blocking ion currents, respectively. We have previously shown a synergistic antiproliferative effect of calcitriol and astemizole in breast cancer cells in vitro, but the effect of this dual therapy in vivo has not been studied.MethodsIn the present study, we explored the combined antineoplastic effect of both drugs in vivo using mice xenografted with the human breast cancer cell line T-47D and a primary breast cancer-derived cell culture (MBCDF). Tumor-bearing athymic female mice were treated with oral astemizole (50 mg/kg/day) and/or intraperitoneal injections of calcitriol (0.03 μg/g body weight twice a week) during 3 weeks. Tumor sizes were measured thrice weekly. For mechanistic insights, we studied EAG1 expression by qPCR and Western blot. The expression of Ki-67 and the relative tumor volume were used as indicators of therapeutic efficacy.ResultsCompared to untreated controls, astemizole and calcitriol significantly reduced, while the coadministration of both drugs further suppressed, tumor growth (P < 0.05). In addition, the combined therapy significantly downregulated tumoral EAG1 and Ki-67 expression.ConclusionsThe concomitant administration of calcitriol and astemizole inhibited tumor growth more efficiently than each drug alone, which may be explained by the blocking of EAG1. These results provide the bases for further studies aimed at testing EAG1-dual targeting in breast cancer tumors expressing both EAG1 and the vitamin D receptor.