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CD38/ADP-ribosyl cyclase: A new role in the regulation of osteoclastic bone resorption.

Sun L, Adebanjo OA, Moonga BS, Corisdeo S, Anandatheerthavarada HK, Biswas G, Arakawa T, Hakeda Y, Koval A, Sodam B, Bevis PJ, Moser AJ, Lai FA, Epstein S, Troen BR, Kumegawa M, Zaidi M - J. Cell Biol. (1999)

Bottom Line: We then examined the effects of CD38 on osteoclast function.IL-6, in turn, enhanced CD38 mRNA expression.Taken together, the results provide compelling evidence for a new role for CD38/ADP-ribosyl cyclase in the control of bone resorption, most likely exerted via cADPr.

View Article: PubMed Central - PubMed

Affiliation: Center for Osteoporosis and Skeletal Aging, Department of Medicine, Medical College of Pennsylvania and Veterans Affairs Medical Center, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
The multifunctional ADP-ribosyl cyclase, CD38, catalyzes the cyclization of NAD(+) to cyclic ADP-ribose (cADPr). The latter gates Ca(2+) release through microsomal membrane-resident ryanodine receptors (RyRs). We first cloned and sequenced full-length CD38 cDNA from a rabbit osteoclast cDNA library. The predicted amino acid sequence displayed 59, 59, and 50% similarity, respectively, to the mouse, rat, and human CD38. In situ RT-PCR revealed intense cytoplasmic staining of osteoclasts, confirming CD38 mRNA expression. Both confocal microscopy and Western blotting confirmed the plasma membrane localization of the CD38 protein. The ADP-ribosyl cyclase activity of osteoclastic CD38 was next demonstrated by its ability to cyclize the NAD(+) surrogate, NGD(+), to its fluorescent derivative cGDP-ribose. We then examined the effects of CD38 on osteoclast function. CD38 activation by an agonist antibody (A10) in the presence of substrate (NAD(+)) triggered a cytosolic Ca(2+) signal. Both ryanodine receptor modulators, ryanodine, and caffeine, markedly attenuated this cytosolic Ca(2+) change. Furthermore, the anti-CD38 agonist antibody expectedly inhibited bone resorption in the pit assay and elevated interleukin-6 (IL-6) secretion. IL-6, in turn, enhanced CD38 mRNA expression. Taken together, the results provide compelling evidence for a new role for CD38/ADP-ribosyl cyclase in the control of bone resorption, most likely exerted via cADPr.

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Histostained osteoclasts following in situ RT-PCR for detection of CD38 mRNA. i shows a negative control from a representative experiment i.e., without added primer. ii and iii represent histostaining for cathepsin K (Cath K) (cell-specific positive control) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (housekeeping gene). Panels iv to vi show CD38 mRNA staining in osteoclasts incubated either in vehicle (iv) or with 10 ng/liter IL6 (v) or 10 μg/liter IL6 (vi). For details and primer sequences refer to Materials and Methods.
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Figure 3: Histostained osteoclasts following in situ RT-PCR for detection of CD38 mRNA. i shows a negative control from a representative experiment i.e., without added primer. ii and iii represent histostaining for cathepsin K (Cath K) (cell-specific positive control) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (housekeeping gene). Panels iv to vi show CD38 mRNA staining in osteoclasts incubated either in vehicle (iv) or with 10 ng/liter IL6 (v) or 10 μg/liter IL6 (vi). For details and primer sequences refer to Materials and Methods.

Mentions: CD38 mRNA expression in isolated single osteoclasts was investigated by in situ RT-PCR cytoimaging using the same primers as used for PCR cloning (above). Fig. 3 shows light micrographs of histostained osteoclasts after RT-PCR. Panel i shows an unstained osteoclast (negative controls) in an experiment in which primers were omitted from the reaction mixture. Panels ii and iii show osteoclasts in which the intense bluish-brown staining represents, respectively, mRNA expression for cathepsin K (cell-specific positive control) or GAPDH (housekeeping gene). Panels iv to vi show intense CD38 mRNA histostaining in osteoclasts that were either incubated with vehicle (iv), 10 ng/liter IL-6 (v), or 10 μg/liter IL-6 (vi).


CD38/ADP-ribosyl cyclase: A new role in the regulation of osteoclastic bone resorption.

Sun L, Adebanjo OA, Moonga BS, Corisdeo S, Anandatheerthavarada HK, Biswas G, Arakawa T, Hakeda Y, Koval A, Sodam B, Bevis PJ, Moser AJ, Lai FA, Epstein S, Troen BR, Kumegawa M, Zaidi M - J. Cell Biol. (1999)

Histostained osteoclasts following in situ RT-PCR for detection of CD38 mRNA. i shows a negative control from a representative experiment i.e., without added primer. ii and iii represent histostaining for cathepsin K (Cath K) (cell-specific positive control) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (housekeeping gene). Panels iv to vi show CD38 mRNA staining in osteoclasts incubated either in vehicle (iv) or with 10 ng/liter IL6 (v) or 10 μg/liter IL6 (vi). For details and primer sequences refer to Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169484&req=5

Figure 3: Histostained osteoclasts following in situ RT-PCR for detection of CD38 mRNA. i shows a negative control from a representative experiment i.e., without added primer. ii and iii represent histostaining for cathepsin K (Cath K) (cell-specific positive control) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (housekeeping gene). Panels iv to vi show CD38 mRNA staining in osteoclasts incubated either in vehicle (iv) or with 10 ng/liter IL6 (v) or 10 μg/liter IL6 (vi). For details and primer sequences refer to Materials and Methods.
Mentions: CD38 mRNA expression in isolated single osteoclasts was investigated by in situ RT-PCR cytoimaging using the same primers as used for PCR cloning (above). Fig. 3 shows light micrographs of histostained osteoclasts after RT-PCR. Panel i shows an unstained osteoclast (negative controls) in an experiment in which primers were omitted from the reaction mixture. Panels ii and iii show osteoclasts in which the intense bluish-brown staining represents, respectively, mRNA expression for cathepsin K (cell-specific positive control) or GAPDH (housekeeping gene). Panels iv to vi show intense CD38 mRNA histostaining in osteoclasts that were either incubated with vehicle (iv), 10 ng/liter IL-6 (v), or 10 μg/liter IL-6 (vi).

Bottom Line: We then examined the effects of CD38 on osteoclast function.IL-6, in turn, enhanced CD38 mRNA expression.Taken together, the results provide compelling evidence for a new role for CD38/ADP-ribosyl cyclase in the control of bone resorption, most likely exerted via cADPr.

View Article: PubMed Central - PubMed

Affiliation: Center for Osteoporosis and Skeletal Aging, Department of Medicine, Medical College of Pennsylvania and Veterans Affairs Medical Center, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
The multifunctional ADP-ribosyl cyclase, CD38, catalyzes the cyclization of NAD(+) to cyclic ADP-ribose (cADPr). The latter gates Ca(2+) release through microsomal membrane-resident ryanodine receptors (RyRs). We first cloned and sequenced full-length CD38 cDNA from a rabbit osteoclast cDNA library. The predicted amino acid sequence displayed 59, 59, and 50% similarity, respectively, to the mouse, rat, and human CD38. In situ RT-PCR revealed intense cytoplasmic staining of osteoclasts, confirming CD38 mRNA expression. Both confocal microscopy and Western blotting confirmed the plasma membrane localization of the CD38 protein. The ADP-ribosyl cyclase activity of osteoclastic CD38 was next demonstrated by its ability to cyclize the NAD(+) surrogate, NGD(+), to its fluorescent derivative cGDP-ribose. We then examined the effects of CD38 on osteoclast function. CD38 activation by an agonist antibody (A10) in the presence of substrate (NAD(+)) triggered a cytosolic Ca(2+) signal. Both ryanodine receptor modulators, ryanodine, and caffeine, markedly attenuated this cytosolic Ca(2+) change. Furthermore, the anti-CD38 agonist antibody expectedly inhibited bone resorption in the pit assay and elevated interleukin-6 (IL-6) secretion. IL-6, in turn, enhanced CD38 mRNA expression. Taken together, the results provide compelling evidence for a new role for CD38/ADP-ribosyl cyclase in the control of bone resorption, most likely exerted via cADPr.

Show MeSH