Two HLA-B27 alleles differently associated with spondylarthritis, B*2709 and B*2705, display similar intracellular trafficking and oligomer formation

OBJECTIVE: To examine whether and to what extent the intracellular trafficking features of HLA-B*2705, which is associated with the development of spondylarthritis (SpA), differ from those of HLA-B*2709 and HLA-B*0702, which are not associated with SpA. METHODS: HeLa cells were transfected with complementary DNA encoding for HLA-B proteins fused to Renilla luciferase or yellow fluorescent protein. The subcellular distribution of properly folded and unfolded/misfolded HLA-B proteins was examined by flow cytometry and confocal microscopy of cells labeled with ME1 and HC-10 antibodies, respectively. HLA-B/HLA-B interactions were monitored in endoplasmic reticulum (ER)- and plasma membrane-enriched subcellular fractions, by bioluminescence resonance energy transfer (BRET). RESULTS: All 3 HLA-B alleles displayed a similar distribution pattern (properly folded heavy chain at the cell surface, unfolded/misfolded proteins only in the cytoplasm). By means of BRET, we provided evidence that both HLA-B*2705 and HLA-B*2709 formed more oligomers in the ER and the plasma membrane than did HLA-B*0702. The propensity of HLA-B*2705 to form oligomers in the ER was partly attributable to residue Cys(67) of the molecule. For all 3 alleles, increased expression of HLA-B proteins was associated with intracytoplasmic accumulation of unfolded/misfolded proteins and intracellular vesicles, probably corresponding to expanded ER-Golgi intermediate compartments, in which these proteins accumulated together with the stress sensor BiP. CONCLUSION: Our results suggest that the difference in disease susceptibility conferred by HLA-B*2705 and HLA-B*2709 cannot be explained by their different propensity to form dimers or misfolded proteins, thus presumably implicating other, still unknown factors.     

Cross-talk between mineralocorticoid and angiotensin II signaling for cardiac remodeling

Experimental and clinical studies show that aldosterone/mineralocorticoid receptor (MR) activation has deleterious effects in the cardiovascular system that may cross-talk with those of angiotensin II (Ang II). This study, using a transgenic mouse model with conditional and cardiomyocyte-restricted overexpression of the human MR, was designed to assess the cardiac consequences of Ang II treatment and cardiomyocyte MR activation. Two-month-old MHCtTA/tetO-hMR double transgenic males (DTg) with conditional, cardiomyocyte-specific human MR expression, and their control littermates were infused with Ang II (200 ng/kg per minute) or vehicle via osmotic minipump. Ang II induced similar increases in systolic blood pressure in control and DTg mice but a greater increase in left ventricle mass/body weight in DTg than in control mice. In DTg mice, Ang II-induced left ventricle hypertrophy and diastolic dysfunction without affecting systolic function, as assessed by echography. These effects were associated with an increase in the expression of collagens and fibronectin, matrix metalloproteinase 2 and matrix metalloproteinase 9 activities, and histological fibrosis. Ang II treatment of DTg mice did not affect inflammation markers, but oxidative stress was substantially increased, as indicated by gp91 expression, apocynin-inhibitable NADPH oxidase activity, and protein carbonylation. These molecular and functional alterations were prevented by pharmacological MR antagonism. Our findings indicate that the effects of Ang II and MR activation in the heart are additive. This observation may be relevant to the clinical use of MR or of combined Ang II type 1 receptor-MR antagonists for hypertrophic cardiomyopathies or for heart failure, particularly when diastolic dysfunction is associated with preserved systolic function.     

Two HLA–B27 alleles differently associated with spondylarthritis,B*2709 and B*2705, display similar intracellular trafficking and oligomer formation

Objective

To examine whether and to what extent the intracellular trafficking features of HLA–B*2705, which is associated with the development of spondylarthritis (SpA), differ from those of HLA–B*2709 and HLA–B*0702, which are not associated with SpA.

Methods

HeLa cells were transfected with complementary DNA encoding for HLA–B proteins fused to Renilla luciferase or yellow fluorescent protein. The subcellular distribution of properly folded and unfolded/misfolded HLA–B proteins was examined by flow cytometry and confocal microscopy of cells labeled with ME1 and HC‐10 antibodies, respectively. HLA–B/HLA–B interactions were monitored in endoplasmic reticulum (ER)– and plasma membrane–enriched subcellular fractions, by bioluminescence resonance energy transfer (BRET).

Results

All 3 HLA–B alleles displayed a similar distribution pattern (properly folded heavy chain at the cell surface, unfolded/misfolded proteins only in the cytoplasm). By means of BRET, we provided evidence that both HLA–B*2705 and HLA–B*2709 formed more oligomers in the ER and the plasma membrane than did HLA–B*0702. The propensity of HLA–B*2705 to form oligomers in the ER was partly attributable to residue Cys⁶⁷ of the molecule. For all 3 alleles, increased expression of HLA–B proteins was associated with intracytoplasmic accumulation of unfolded/misfolded proteins and intracellular vesicles, probably corresponding to expanded ER–Golgi intermediate compartments, in which these proteins accumulated together with the stress sensor BiP.

Conclusion

Our results suggest that the difference in disease susceptibility conferred by HLA–B*2705 and HLA–B*2709 cannot be explained by their different propensity to form dimers or misfolded proteins, thus presumably implicating other, still unknown factors.