Endoplasmic reticulum (ER) stress is associated with diabetic nephropathy (DN), but its pathophysiological relevance and the mechanisms that compromise adaptive ER signalling in podocytes remain unknown. Here we show that nuclear translocation of the transcription factor spliced X-box binding protein-1 (sXBP1) is selectively impaired in DN, inducing activating transcription factor-6 (ATF6) and C/EBP homology protein (CHOP). Podocyte-specific genetic ablation of XBP1 or inducible expression of ATF6 in mice aggravate DN. sXBP1 lies downstream of insulin signalling and attenuating podocyte insulin signalling by genetic ablation of the insulin receptor or the regulatory subunits phosphatidylinositol 3-kinase (PI3K) p85α or p85β impairs sXBP1 nuclear translocation and exacerbates DN. Corroborating our findings from murine DN, the interaction of sXBP1 with p85α and p85β is markedly impaired in the glomerular compartment of human DN. Thus, signalling via the insulin receptor, p85, and XBP1 maintains podocyte homeostasis, while disruption of this pathway impairs podocyte function in DN.

TFF3 is a secretory peptide belonging to the trefoil factor family with a predicted size of 59 amino acid residues containing seven cysteine residues. It is predominantly expressed in intestinal goblet cells where it plays a key role in mucosal regeneration and repair processes. In the course of these studies, human colonic TFF3 was shown to exist mainly as a high molecular weight heteromer. Purification of this heteromer and characterization by LC-ESI-MS/MS analysis identified the IgG Fc binding protein (FCGBP) as the disulfide-linked partner protein of TFF3. FCGBP is a constituent of intestinal mucus secreted by goblet cells. Furthermore, low amounts of TFF3/monomer and only little TFF3/dimer were detected in human colonic extracts. Here, we show that these TFF3 forms can be released from the purified TFF3-FCGBP heteromer complex in vitro by reduction with hydrogen sulfide (H(2)S). Such a mechanism would be in line with the high H(2)S concentrations reported to occur in the lumen of the colon. Of special note, this points to intestinal mucus as a reservoir for a biologically active peptide. Also proteolytic processing of FCGBP was observed which is in line with multiple autocatalytic cleavages as proposed earlier by Johansson et al. (J. Proteome Res. 2009 , 8 , 3549 - 3557).

Recently, we have shown that the new G-protein-coupled estrogen receptor GPR30 plays an important role in the development of tamoxifen resistance in vitro. This study was undertaken to evaluate the correlation between GPR30 and tamoxifen resistance in breast cancer patients. GPR30 protein expression was evaluated by immunohistochemical analysis in 323 patients with primary operable breast cancer. The association between GPR30 expression and tamoxifen resistance was confirmed in a second cohort of 103 patients treated only with tamoxifen. Additionally, we evaluated GPR30 expression in 33 primary tumors and in recurrent tumors from the same patients. GPR30 expression was detected in 56.7% of the breast cancer specimens investigated and it correlated with overexpression of HER-2 (P = 0.021), EGFR (P = 0.024) and lymph node status (P = 0.047). In a first cohort, survival analysis showed that GPR30 was negatively correlated with relapse-free survival (RFS) only in patients treated with tamoxifen (tamoxifen with or without chemotherapy). GPR30 expression was associated with shorter RFS (HR = 1.768; 95% CI, 1.156-2.703; P = 0.009). In a subset of patients treated only with tamoxifen, multivariate analysis revealed that GPR30 expression is an independent unfavorable factor for RFS (HR = 4.440; 95% CI, 1.408-13.997; P = 0.011). In contrast, GPR30 tended to be a favorable factor regarding RFS in patients who did not receive tamoxifen. In 33 paired biopsies obtained before and after adjuvant therapy, GPR30 expression significantly increased only under tamoxifen treatment (P = 0.001). GPR30 expression in breast cancer independently predicts a poor RFS in patients treated with tamoxifen.

Tamoxifen is the most frequently used anti-hormonal drug for treatment of women with hormone-dependent breast cancer. The aim of this study is to investigate the mechanism of tamoxifen resistance and the impact of the new estrogen G-protein coupled receptor (GPR30). MCF-7 cells were continuously exposed to tamoxifen for 6 months to induce resistance to the inhibitory effect of tamoxifen. These tamoxifen-resistant cells (TAM-R) exhibited enhanced sensitivity to 17-ss-estradiol and GPR30 agonist, G1, when compared to the parental cells. In TAM-R cells, tamoxifen was able to stimulate the cell growth and MAPK phosphorylation. These effects were abolished by EGFR inhibitor AG1478, GPR30 anti-sense oligonucleotide, and the selective c-Src inhibitor PP2. Only EGFR basal expression was slightly elevated in the TAM-R cells, whereas GPR30 expression and the basal phosphorylation of Akt and MAPK remained unchanged when compared to the parental cells. Interestingly, estrogen treatment significantly increased GPR30 translocation to the cell surface, which was stronger in TAM-R cells. Continuous treatment of MCF-7 cells with GPR30 agonist G1 mimics the long-term treatment with tamoxifen and increases drastically its agonistic activity. This data suggests the important role of GPR30/EGFR receptor signaling in the development of tamoxifen resistance. The inhibition of this pathway is a valid option to improve anti-hormone response in breast cancer.