Limits...
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.

Show MeSH

Related in: MedlinePlus

ADP-ribosyl cyclase activity of CD38 in isolated osteoclast plasma membranes. The conversion of the NAD+ surrogate, NGD+, to the nonhydrolyzable fluorescent product, cyclic GDP-ribose (cGDPr) was assessed fluorimetrically (see Materials and Methods for details). Results are expressed as a mean (± SEM) cGDPr formed in nmoles/min/mg isolated plasma membrane protein without treatment (basal) or in the presence of nonimmune mouse serum (IgG5) (NIMS), an antagonist anti-CD38 antibody (Ab; Sigma Chemical Co., 1:500) or NAD+ (400 μM). The asterisks represent significant differences compared with basal (P < 0.05).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2169484&req=5

Figure 7: ADP-ribosyl cyclase activity of CD38 in isolated osteoclast plasma membranes. The conversion of the NAD+ surrogate, NGD+, to the nonhydrolyzable fluorescent product, cyclic GDP-ribose (cGDPr) was assessed fluorimetrically (see Materials and Methods for details). Results are expressed as a mean (± SEM) cGDPr formed in nmoles/min/mg isolated plasma membrane protein without treatment (basal) or in the presence of nonimmune mouse serum (IgG5) (NIMS), an antagonist anti-CD38 antibody (Ab; Sigma Chemical Co., 1:500) or NAD+ (400 μM). The asterisks represent significant differences compared with basal (P < 0.05).

Mentions: CD38 is not only an ADP-ribosyl cyclase that converts NAD+ to cADPr, but is also an ADP hydrolase converting active cADPr to inactive ADP-ribose. It is difficult to separate the two reactions that proceed simultaneously. We have therefore used an assay that monitors cyclization of NGD+ to cGDPr, a nonhydrolyzable cADPr surrogate. Thus, the rate of cGDPr formation, in the absence of its breakdown, will more accurately reflect the ADP-ribosyl cyclase activity of CD38. Furthermore, cGDPr is a fluorescent compound that can be quantitated by fluorimetry. We found that osteoclast plasma membranes synthesized cGDPr at a rate of 4.3 nmoles/min/mg protein (Fig. 7). The anti-CD38 antagonist antibody (Sigma Chemical Co.) inhibited cGDPr formation significantly, thus attributing the observed ADP ribosyl cyclase activity to CD38. Enzyme activity was also inhibited significantly by addition of NAD+ (400 μM), indicating a possible competition between the two nucleotides. Similar results were obtained from postnuclear membranes prepared MC3T3-E1 osteoblasts (not shown).


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)

ADP-ribosyl cyclase activity of CD38 in isolated osteoclast plasma membranes. The conversion of the NAD+ surrogate, NGD+, to the nonhydrolyzable fluorescent product, cyclic GDP-ribose (cGDPr) was assessed fluorimetrically (see Materials and Methods for details). Results are expressed as a mean (± SEM) cGDPr formed in nmoles/min/mg isolated plasma membrane protein without treatment (basal) or in the presence of nonimmune mouse serum (IgG5) (NIMS), an antagonist anti-CD38 antibody (Ab; Sigma Chemical Co., 1:500) or NAD+ (400 μM). The asterisks represent significant differences compared with basal (P < 0.05).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: ADP-ribosyl cyclase activity of CD38 in isolated osteoclast plasma membranes. The conversion of the NAD+ surrogate, NGD+, to the nonhydrolyzable fluorescent product, cyclic GDP-ribose (cGDPr) was assessed fluorimetrically (see Materials and Methods for details). Results are expressed as a mean (± SEM) cGDPr formed in nmoles/min/mg isolated plasma membrane protein without treatment (basal) or in the presence of nonimmune mouse serum (IgG5) (NIMS), an antagonist anti-CD38 antibody (Ab; Sigma Chemical Co., 1:500) or NAD+ (400 μM). The asterisks represent significant differences compared with basal (P < 0.05).
Mentions: CD38 is not only an ADP-ribosyl cyclase that converts NAD+ to cADPr, but is also an ADP hydrolase converting active cADPr to inactive ADP-ribose. It is difficult to separate the two reactions that proceed simultaneously. We have therefore used an assay that monitors cyclization of NGD+ to cGDPr, a nonhydrolyzable cADPr surrogate. Thus, the rate of cGDPr formation, in the absence of its breakdown, will more accurately reflect the ADP-ribosyl cyclase activity of CD38. Furthermore, cGDPr is a fluorescent compound that can be quantitated by fluorimetry. We found that osteoclast plasma membranes synthesized cGDPr at a rate of 4.3 nmoles/min/mg protein (Fig. 7). The anti-CD38 antagonist antibody (Sigma Chemical Co.) inhibited cGDPr formation significantly, thus attributing the observed ADP ribosyl cyclase activity to CD38. Enzyme activity was also inhibited significantly by addition of NAD+ (400 μM), indicating a possible competition between the two nucleotides. Similar results were obtained from postnuclear membranes prepared MC3T3-E1 osteoblasts (not shown).

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