Expression of sarco/endoplasmic reticular Ca(2+)-ATPase in human lens epithelial cells and cultured human lens epithelial B-3 cells.
OBJECTIVE
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.


CONCLUSIONS
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.
Liu
L
L
University of Louisville, Department of Ophthalmology and Visual Sciences, Louisville, KY, USA.
Bian
L
L
Borchman
D
D
Paterson
C A
CA
D000818:Animals; D015153:Blotting, Western; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D004847:Epithelial Cells / Q000201:enzymology; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007908:Lens, Crystalline / Q000166:cytology / Q000378:metabolism*; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D007527:Isoenzymes; D012333:RNA, Messenger; D000252:Calcium-Transporting ATPases