Expression of sarco/endoplasmic reticular Ca2+-ATPase in human lens epithelial cells and cultured human lens epithelial B-3 cells

Abstract

PURPOSE. Sarco/Endoplasmic Reticulum Ca 2+ -ATPase (SER-CAs) isoforms, SERCA2b (100Kd) and SERCA3 (97Kd), are co-expressed in some non-muscle tissues. Both types of SERCAs play important roles in Ca 2+ regulation in non-muscle tissues such as brain and platelets. We tested whether these two SERCAs are present in human lens epithelial cells, human lens epithelial B-3 cells (HLE B-3 cells) and rat lens epithelial cells providing a basis for further studies the role of SERCAs in cataract formation. METHODS. Lens epithelial cells from rat lens, human lens and cultured human lens epithelial B-3 cell line (HLE B-3) were used to extract mRNA and membrane proteins. Reverse transcription polymerase chain reaction (RT-PCR) was employed to detect SERCA3 and SERCA2b mRNA using SERCA2b and SERCA3 primers. Western blotting was used to detect SERCA3 and SERCA2b proteins using anti-SERCA2b and 3 antibodies. RESULTS. A 270 bp fragment was amplified with SERCA2b primers and a 186 bp fragment amplified with SERCA3 primer in human lens epithelial cells, human lens epithelial B-3 cells and rat lens epithelial cells. Both fragments are the expected SERCA2b and SERCA3 products. The sequences of these two fragments in human lens epithelial cells are 100% and 98% homologous to the published partial SERCA2b and SERCA3 mRNA sequence, respectively. The amino acid sequences translated from both PCR products are 100% homologous to the published SERCA2b and SERCA3 sequences in GeneBank. Western blotting confirmed that the expected 100 KDa and a 97 KDa proteins are recognized by anti-SERCA2b and anti-SERCA3 antibodies. CONCLUSION. Sarco/Endoplasmic Reticulum Ca 2+ -ATPase (SERCAs) isoforms 2b and 3 mRNAs are expressed in human lens epithelial cells, the cultured human lens epithelial B-3 cell line (HLE B-3) and rat lens epithelial cells. SERCA2b and SERCA3 may play an important role in the regulation of calcium homeostasis in lens epithelial cells.