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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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Semiquantitative representation in frequency histograms of intensity score after in situ RT-PCR for CD38 mRNA in osteoclasts incubated either without primer (i) or with CD38 primers without IL-6 treatment (ii) or with IL-6 treatment (10 ng/l, iii, or 10 μg/l, iv). Staining intensity was graded as described in Materials and Methods by an independent blinded observer who scored the intensity from zero (no staining) to 4 (intense staining) in three experiments. The data were analyzed statistically for skewness and shifts were considered significant if P < 0.05.
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Figure 4: Semiquantitative representation in frequency histograms of intensity score after in situ RT-PCR for CD38 mRNA in osteoclasts incubated either without primer (i) or with CD38 primers without IL-6 treatment (ii) or with IL-6 treatment (10 ng/l, iii, or 10 μg/l, iv). Staining intensity was graded as described in Materials and Methods by an independent blinded observer who scored the intensity from zero (no staining) to 4 (intense staining) in three experiments. The data were analyzed statistically for skewness and shifts were considered significant if P < 0.05.

Mentions: We then performed an analysis of the staining intensity using a blinded observer. Osteoclasts were scored on a scale from 0 to 4 (no staining to intense staining, see legend to Fig. 4). The results from three experiments were plotted as a frequency histogram. This allowed us to determine the proportion of cells that lay in a certain intensity range. A similar analysis has been used by us previously (Adebanjo et al. 1998) to demonstrate the effects of Ca2+ on IL-6 and IL-6 receptor mRNA expression. Notably, GAPDH, a housekeeping gene, was used to control for the effects of IL-6 on CD38 expression. Also note, that, as previously, the few retracted osteoclasts were discarded from the analysis to prevent a biased intensity assessment.


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)

Semiquantitative representation in frequency histograms of intensity score after in situ RT-PCR for CD38 mRNA in osteoclasts incubated either without primer (i) or with CD38 primers without IL-6 treatment (ii) or with IL-6 treatment (10 ng/l, iii, or 10 μg/l, iv). Staining intensity was graded as described in Materials and Methods by an independent blinded observer who scored the intensity from zero (no staining) to 4 (intense staining) in three experiments. The data were analyzed statistically for skewness and shifts were considered significant if P < 0.05.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Semiquantitative representation in frequency histograms of intensity score after in situ RT-PCR for CD38 mRNA in osteoclasts incubated either without primer (i) or with CD38 primers without IL-6 treatment (ii) or with IL-6 treatment (10 ng/l, iii, or 10 μg/l, iv). Staining intensity was graded as described in Materials and Methods by an independent blinded observer who scored the intensity from zero (no staining) to 4 (intense staining) in three experiments. The data were analyzed statistically for skewness and shifts were considered significant if P < 0.05.
Mentions: We then performed an analysis of the staining intensity using a blinded observer. Osteoclasts were scored on a scale from 0 to 4 (no staining to intense staining, see legend to Fig. 4). The results from three experiments were plotted as a frequency histogram. This allowed us to determine the proportion of cells that lay in a certain intensity range. A similar analysis has been used by us previously (Adebanjo et al. 1998) to demonstrate the effects of Ca2+ on IL-6 and IL-6 receptor mRNA expression. Notably, GAPDH, a housekeeping gene, was used to control for the effects of IL-6 on CD38 expression. Also note, that, as previously, the few retracted osteoclasts were discarded from the analysis to prevent a biased intensity assessment.

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