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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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Related in: MedlinePlus

Western blotting after SDS-PAGE of osteoclast plasma membranes (OC-PM) (30 μg protein) and osteoblast postnuclear membranes (OB-PNM) (50 μg protein) using an antagonist anti-CD38 antibody (Sigma Chemical Co.). We identified 46-kD bands with an additional lower molecular weight band in OC-PMs. The osteoclast preparations were >99% pure as assessed by TRAP staining (see Materials and Methods).
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Figure 6: Western blotting after SDS-PAGE of osteoclast plasma membranes (OC-PM) (30 μg protein) and osteoblast postnuclear membranes (OB-PNM) (50 μg protein) using an antagonist anti-CD38 antibody (Sigma Chemical Co.). We identified 46-kD bands with an additional lower molecular weight band in OC-PMs. The osteoclast preparations were >99% pure as assessed by TRAP staining (see Materials and Methods).

Mentions: We further confirmed that the CD38 protein was present in isolated osteoclast plasma membranes by Western blotting using a different antagonist anti-CD38 antibody (Sigma Chemical Co.). A ∼46 kD band was observed when plasma membranes purified by sucrose gradient centrifugation were electrophoresed and immunoblotted (Fig. 6). A further, significantly weaker, band of a smaller molecular weight (∼39 kD) was also seen; this may represent a degradation product, but we are unclear of its identity. The latter band was not obvious when post-nuclear membranes from osteoblastic MC3T3-E1 cells were similarly immunoblotted. Note that the purity of the osteoclastic preparations was >99% based on TRAP staining (see Materials and Methods).


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)

Western blotting after SDS-PAGE of osteoclast plasma membranes (OC-PM) (30 μg protein) and osteoblast postnuclear membranes (OB-PNM) (50 μg protein) using an antagonist anti-CD38 antibody (Sigma Chemical Co.). We identified 46-kD bands with an additional lower molecular weight band in OC-PMs. The osteoclast preparations were >99% pure as assessed by TRAP staining (see Materials and Methods).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: Western blotting after SDS-PAGE of osteoclast plasma membranes (OC-PM) (30 μg protein) and osteoblast postnuclear membranes (OB-PNM) (50 μg protein) using an antagonist anti-CD38 antibody (Sigma Chemical Co.). We identified 46-kD bands with an additional lower molecular weight band in OC-PMs. The osteoclast preparations were >99% pure as assessed by TRAP staining (see Materials and Methods).
Mentions: We further confirmed that the CD38 protein was present in isolated osteoclast plasma membranes by Western blotting using a different antagonist anti-CD38 antibody (Sigma Chemical Co.). A ∼46 kD band was observed when plasma membranes purified by sucrose gradient centrifugation were electrophoresed and immunoblotted (Fig. 6). A further, significantly weaker, band of a smaller molecular weight (∼39 kD) was also seen; this may represent a degradation product, but we are unclear of its identity. The latter band was not obvious when post-nuclear membranes from osteoblastic MC3T3-E1 cells were similarly immunoblotted. Note that the purity of the osteoclastic preparations was >99% based on TRAP staining (see Materials and Methods).

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
Related in: MedlinePlus