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
Effect of NAD+ (NAD+, concentrations as shown) on the mean change (Δ) in cytosolic [Ca2+] (nM) in fura 2–loaded single osteoclasts under various experimental conditions. (A) Osteoclasts were preincubated with either vehicle or the anti-CD38 agonist antibody, A10 (1:500) in Medium 199-Hanks ([Ca2+] = 1.25 mM). (B) Osteoclasts were pretreated for several min with the microsomal membrane Ca2+-ATPase inhibitor, thapsigargin (4 μM), or with the cell-permeant ryanodine receptor modulators, ryanodine (4 μM) or caffeine (4 μM). (C) Osteoclasts were pretreated for 15 min with the antagonist antibody (Sigma Chemical Co.) (1:500) (Sig). Cytosolic Δ [Ca2+] was calculated in each case by subtracting the basal from peak cytosolic [Ca2+]. Asterisks indicate P < 0.05 (n = 6 per group).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2169484&req=5

Figure 8: Effect of NAD+ (NAD+, concentrations as shown) on the mean change (Δ) in cytosolic [Ca2+] (nM) in fura 2–loaded single osteoclasts under various experimental conditions. (A) Osteoclasts were preincubated with either vehicle or the anti-CD38 agonist antibody, A10 (1:500) in Medium 199-Hanks ([Ca2+] = 1.25 mM). (B) Osteoclasts were pretreated for several min with the microsomal membrane Ca2+-ATPase inhibitor, thapsigargin (4 μM), or with the cell-permeant ryanodine receptor modulators, ryanodine (4 μM) or caffeine (4 μM). (C) Osteoclasts were pretreated for 15 min with the antagonist antibody (Sigma Chemical Co.) (1:500) (Sig). Cytosolic Δ [Ca2+] was calculated in each case by subtracting the basal from peak cytosolic [Ca2+]. Asterisks indicate P < 0.05 (n = 6 per group).

Mentions: Having established the presence of CD38 in the osteoclast plasma membrane, we next investigated the effects of its activation by the agonist anti-CD38 antibody. Thus, we measured changes in cytosolic [Ca2+] in response to application of NAD+ (substrate) in the presence of the agonist antibody. Expectedly, only in the presence of the antibody (1:500), did 1 mM NAD+ trigger a cytosolic [Ca2+] elevation (Fig. 8 A). This result was consistent with an activated CD38/ADP-ribosyl cyclase that catalyses cADPr generation from NAD+. In separate experiments, the anti-CD38 antibody itself, in the absence of NAD+, did not elevate cytosolic [Ca2+], indicating that the substrate, NAD+, was necessary for CD38-induced Ca2+ signaling (not shown). Finally, 1 mM NAD+ failed to trigger a cytosolic Ca2+ signal in the presence of the control anti-RyR antibody, Ab34, further confirming response specificity.


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)

Effect of NAD+ (NAD+, concentrations as shown) on the mean change (Δ) in cytosolic [Ca2+] (nM) in fura 2–loaded single osteoclasts under various experimental conditions. (A) Osteoclasts were preincubated with either vehicle or the anti-CD38 agonist antibody, A10 (1:500) in Medium 199-Hanks ([Ca2+] = 1.25 mM). (B) Osteoclasts were pretreated for several min with the microsomal membrane Ca2+-ATPase inhibitor, thapsigargin (4 μM), or with the cell-permeant ryanodine receptor modulators, ryanodine (4 μM) or caffeine (4 μM). (C) Osteoclasts were pretreated for 15 min with the antagonist antibody (Sigma Chemical Co.) (1:500) (Sig). Cytosolic Δ [Ca2+] was calculated in each case by subtracting the basal from peak cytosolic [Ca2+]. Asterisks indicate P < 0.05 (n = 6 per group).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: Effect of NAD+ (NAD+, concentrations as shown) on the mean change (Δ) in cytosolic [Ca2+] (nM) in fura 2–loaded single osteoclasts under various experimental conditions. (A) Osteoclasts were preincubated with either vehicle or the anti-CD38 agonist antibody, A10 (1:500) in Medium 199-Hanks ([Ca2+] = 1.25 mM). (B) Osteoclasts were pretreated for several min with the microsomal membrane Ca2+-ATPase inhibitor, thapsigargin (4 μM), or with the cell-permeant ryanodine receptor modulators, ryanodine (4 μM) or caffeine (4 μM). (C) Osteoclasts were pretreated for 15 min with the antagonist antibody (Sigma Chemical Co.) (1:500) (Sig). Cytosolic Δ [Ca2+] was calculated in each case by subtracting the basal from peak cytosolic [Ca2+]. Asterisks indicate P < 0.05 (n = 6 per group).
Mentions: Having established the presence of CD38 in the osteoclast plasma membrane, we next investigated the effects of its activation by the agonist anti-CD38 antibody. Thus, we measured changes in cytosolic [Ca2+] in response to application of NAD+ (substrate) in the presence of the agonist antibody. Expectedly, only in the presence of the antibody (1:500), did 1 mM NAD+ trigger a cytosolic [Ca2+] elevation (Fig. 8 A). This result was consistent with an activated CD38/ADP-ribosyl cyclase that catalyses cADPr generation from NAD+. In separate experiments, the anti-CD38 antibody itself, in the absence of NAD+, did not elevate cytosolic [Ca2+], indicating that the substrate, NAD+, was necessary for CD38-induced Ca2+ signaling (not shown). Finally, 1 mM NAD+ failed to trigger a cytosolic Ca2+ signal in the presence of the control anti-RyR antibody, Ab34, further confirming response specificity.

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