Cellular distribution and regulation of NHE-1 isoform of the NA-H exchanger in the avian osteoclast.
The sodium-hydrogen exchanger (NHE) has been implicated in bone resorption by osteoclasts. We have studied expression of NHE-1, an isoform of the NHE, in chicken bone marrow mononuclear phagocyte precursors during differentiation into the osteoclast phenotype in culture. A monoclonal anti-body raised against the carboxy-terminus of NHE-1 detected the presence of a 100 kDa protein (similar to the mammalian form of NHE-1) in the osteoclasts. Laser scanning confocal microscopy revealed association with the alpha(v)beta(3) integrin and focal adhesion kinase (pp(125)FAK) at the basolateral membrane (BLM) of the osteoclast in addition to a more generalized cellular distribution. A fragment of avian NHE-1 cDNA was obtained by polymerase chain reaction cloning, and it was used to characterize expression of NHE-1 transcripts in cultured chicken osteoclast precursors. The avian NHE-1 message was a 3.9 kB band on Northern analysis, which differed from the mammalian message. Retinoic acid (RA) elicited an increase in the steady-state intracellular pH (pH(1)) from 6.87 to 7.10 in the absence of bicarbonate and was inhibited by ethylisopropylamiloride, an inhibitor of Na-H exchange. Using ribonuclease protection assays, we found that NHE-1 transcripts are induced as cells differentiate in vitro and in response to 13-cis-RA. Western blot analysis indicated that protein levels also increased in response to 13-cis-RA. Our results demonstrate expression of NHE-1 in avian osteoclasts with a complex cellular distribution in culture, and NHE-1 expression is induced as cells differentiate into mature osteoclasts in response to 13-cis-RA.
Gupta
A
A
Renal Division, Jewish Hospital of St. Louis, MO, USA.
Edwards
J C
JC
Hruska
K A
KA
D000595:Amino Acid Sequence; D000818:Animals; D001853:Bone Marrow / Q000378:metabolism*; D001854:Bone Marrow Cells; D002118:Calcium / Q000502:physiology*; D002454:Cell Differentiation / Q000502:physiology; D002478:Cells, Cultured; D002645:Chickens; D005786:Gene Expression Regulation / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D015474:Isotretinoin / Q000494:pharmacology; D008969:Molecular Sequence Data; D010010:Osteoclasts / Q000187:drug effects / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism*
D012333:RNA, Messenger; D017923:Sodium-Hydrogen Exchangers; C069139:growth factor-activatable Na-H exchanger NHE-1; D015474:Isotretinoin; D002118:Calcium