CD38 is required for priming by TNF- : a mechanism for extracellular coordinatio - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-22 09:41 |只看该作者 |倒序浏览 |打印

作者：Jameel Iqbal and Mone Zaidi作者单位：Mount Sinai Bone Program, Mount Sinai School of Medicine, New York, New York 5 N! ?: a* w  W5 @" M

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      【摘要】
   Cytokines are protein messengers that act to modulate the differentiation or activation of their target cells. Bone marrow macrophages can become activated tissue macrophages, dendritic cells, or osteoclasts depending on to which cytokines they are exposed. However, one cytokine can often induce divergent outcomes, suggesting that other signals are needed to establish the specificity of the result. We hypothesize that these signals may derive from the local environment and serve to prime cells to respond toward a specific outcome. Here, it is shown that the cytokine TNF- is capable of affecting the fate of macrophages by upregulating the NADase CD38. CD38 upregulation primes macrophages, such that signals induced by inflammatory stimuli are augmented, while those leading to osteoclast formation are inhibited. We show that TNF- -induced CD38 expression negatively affects the expression of osteoclast markers, while it enhances inflammatory gene expression by decreasing ERK1/2 phosphorylation and increasing NF- B activation. Furthermore, it is shown that CD38 may reduce osteoclastogenesis and increase inflammatory gene induction by decreasing cellular histone deacetylase activity. These results provide a demonstration of how a cytokine can prime cells to differentiate toward a certain lineage or acquire enhanced activation characteristics. Since CD38 is an ectoenzyme, we suggest that the modulation of extracellular NAD metabolism likely serves as a unique mechanism to coordinate the fate of cells within a local environment. 1 e4 X+ A( D% d! p& {, F' ~/ S
      【关键词】 osteoclast LPS inflammation IL cfos cRel differentiation
   IN THEIR ROLE AS PROTEIN MESSENGERS that convey information to neighboring cells, cytokines modulate several signaling pathways to determine specific outcomes. However, how two different cytokines produce unique outcomes when they activate the same signaling pathways remains unclear. Different outcomes may in part be due to subtle differences in the amount or time of activation of each pathway. For example, we have shown recently that TNF- but not RANK-L upregulates the enzyme CD38, despite the fact that the two cytokines initially induce identical signals ( 10 ). We have attributed this differential upregulation to subtleties in JNK, NF- B, and PKC activation ( 10 ).* x" p% n$ y2 _7 v- \

Even more intriguing is how a single cytokine can produce unique outcomes. For example, the cytokine RANK-L is necessary for both osteoclast and lymph node formation ( 13, 14 ). One possible explanation for this divergence in outcome is that the cellular environment within which the cells are exposed to the cytokine may be different and thus critically influence the result. This influence likely occurs through "priming" of the cells, such that they are predisposed to respond in a certain way. In many ways, these priming cues are critical for generating the proper outcome; for example, an osteoclast only releases acid and proteolytic enzymes when it is attached to a matrix, where integrin signaling acts to prime the cells for RANK-L-induced resorption.

Priming of adaptive immune responses, as well as enhancement of innate immunity, occurs through the production of cytokines by cells of the innate immune system ( 31 ). Exposure of T lymphocytes to the proinflammatory cytokine TNF- allows rapid production of IL-2 and interferon- mRNA, enabling cytokine-primed cells to achieve quicker and higher levels of these cytokines in response to a subsequent exposure ( 4 ). Priming by TNF- occurs partly through alterations in signal transduction pathways, such as NF- B ( 4 ), and partly through increases in the production of proinflammatory autocrine/paracrine stimulants, such as interferon- ( 8 ).
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Recent evidence suggests that changes in NAD metabolism can also alter cell fate. For example, increases in the NAD metabolite nicotinamide can decrease histone deacetylase (HDAC) activity and augment NF- B activation ( 7, 35 ). We have recently shown that TNF- affects cellular NAD metabolism by upregulating the ectoenzyme CD38 ( 11 ). CD38 is a NADase but can also act as an ADP-ribosyl cyclase, cleaving NAD into cyclic ADP-ribose. Both ADP-ribose and cADP-ribose can modulate Ca 2  signaling by enhancing the opening potential of TRP channels or ryanodine receptor-gated channels, respectively ( 9, 21 ). Through this mechanism, CD38 is necessary for the generation of Ca 2  signals downstream of certain G protein-coupled receptors ( 24, 25 ).

In this investigation, we sought to determine whether parts of the priming actions of TNF- were mediated through CD38 upregulation. Here, we report how TNF- -induced upregulation of the NADase CD38 can impact the fate of primary murine macrophages. We demonstrate that TNF- primes macrophages through CD38 upregulation, which serves to decrease ERK1/2 activation and HDAC activity, and to increase NF- B activation. Through these mechanisms, CD38 dramatically enhances sensitivity to inflammatory stimuli and decreases the propensity of macrophages to differentiate into osteoclasts.' `5 K! j* A  ~/ [( Z* c

METHODS* \0 L8 b3 Q( e% Y2 [5 Z

Cell preparation. To obtain total bone marrow, mice were killed by an institutionally approved protocols and both femurs and tibea were surgically extracted and their bone marrow was flushed. The bone marrow pellet was resuspended in -MEM (Invitrogen, Carlsbad, CA) containing 10% FBS (Select USA stock, Invitrogen) and 1% penicillin/streptomycin. Further information can be obtained elsewhere ( 10, 11 )., V5 F! j  z) ^; a, ?2 F/ k
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Osteoclast formation. Recombinant murine M-CSF (5 ng/ml, R&D Systems, Minneapolis, MN) was added, and the cells were incubated for 1 day. Nonadherent cells were collected and layered over 6 ml of Ficoll-Hypaque (Amersham/GE Health Sciences, Piscataway, NJ) for density centrifugation. The cells from the interface layer were collected and resuspended in -MEM containing 10% FBS and 1% P/S. Cells were plated at 3 x 10 4 /well in a 96-well plate with 30 ng/ml M-CSF and various concentrations of TNF- or RANK-L (R&D Systems). After 5 days, the cells were stained according to BD Biosciences Technical Bulletin 445. Further details can be found elsewhere ( 10 ).; d. O% w3 f1 h  m9 B$ F. r2 N
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Western blotting. Cells were washed twice with ice-cold 1 x PBS and then scraped using 1 x lysis buffer (Cell Signaling Technologies, Danvers, MA). Cells were then sonicated four times on ice. After centrifugation, the protein concentration of the supernatent was measured, and equal amounts of extracts were loaded onto Novex NuPage 10% Bis-Tris gels (Invitrogen). The gels were electrophoresed in a Novex chamber according to the manufacturer's recommendations (Invitrogen). The gels were then transferred to nitrocellulose membranes using the Novex tank transfer apparatus. After transfer, the membranes were blocked, probed with the antibodies indicated below (see figure legends; from either Santa Cruz Biotechnology, Santa Cruz, CA or Cell Signaling Technologies), and chemiluminescence was performed using the Western Breeze blotting kit (Invitrogen). Western blotting was performed twice.

Cellular HDAC assay. HDAC activity was measured by using the Flur de Lys HDAC kit (AK-500 from Biomol, Plymouth Meeting, PA). Briefly, equal numbers of wild-type and CD38 -/- cells were grown in quadruplicate, and cells were treated with 40 ng/ml TNF- or control for 12 h before the addition of the HDAC substrate. At 0, 2, and 4 h after addition of the substrate, the samples were assayed for the amount of deacetylated substrate by measuring the fluorescence in a fluorimeter (FMax, Molecular Devices, Sunnyvale, CA). HDAC assays were performed twice.

Murine IL-12 ELISA. Whole blood from wild-type and CD38 -/- mice was collected in EDTA-containing tubes to prevent coagulation. The levels of IL-12 in the plasma were measured using an IL-12 ELISA kit (cat. no. 431605, BioLegend, San Diego, CA) following the manufacturer's directions and including controls. The IL-12 assay was performed twice.) z5 I) Y* k  ?, i! C% E! j) @
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Chromatin immunoprecipitation and real-time PCR assays. Both chromatin immunoprecipitation (ChIP) assays and real-time PCR experiments were carried out as we have recently described in detail elsewhere ( 10, 11 ).5 b( l  o% ~  g# }
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Statistics. Group means and SEs were calculated and plotted using Excel 2004 for Mac and subsequently exported to Adobe Illustrator CS2. Student's t -test, using Excel, compared groups for differences deemed significant at P
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Fig. 2. CD38 negatively regulates the expression of osteoclast markers in response to TNF- or RANK-L. Ficoll-purified bone marrow cells were treated with either 60 ng/ml murine RANK-L alone or 40 ng/ml TNF- alone, and the amount of mRNA expression of the osteoclast markers TRAP and cathepsin K was determined by real-time PCR using murine 2-microglobulin as a control gene. The levels of the proosteoclastogenic transcription factor c-fos were also determined relative to 2-microglobulin. The expression levels of CD157 were not different between wild-type and CD38 -/- cells, confirming that CD157 was not upregulated to compensate for the loss of CD38. P

RESULTS* u, G1 H/ ?( @% f
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Absence of CD38 enhances the osteoclastogenic potential of bone marrow macrophages to TNF-. We have previously shown that osteoclast formation is enhanced in bone marrow cultures from CD38-deficient animals ( 32 ). Recently, we have discovered that this enhancement was due in part to the absence of CD38 on contaminating lymphocytes (Iqbal J and Zaidi M, unpublished observations). Thus the osteoclastogenic effect of CD38 expression on osteoclast precursors themselves (referred to hereafter as bone marrow macrophages) remains unclear.

The inflammatory cytokine TNF- induces osteoclastogenesis in vitro at very high doses of TNF- or with "help" provided by other cytokines such as TGF- ( 20 ). We have recently shown that TNF- can greatly increase CD38 expression on bone marrow macrophages ( 10 ). Because CD38 expression negatively modulates RANK-L-induced osteoclast formation, we hypothesized that TNF- -induced CD38 expression on these cells might also negatively impacts TNF- -induced osteoclastogenesis. In other words, we hypothesized that TNF- -induced CD38 upregulation may prevent TNF- from being able to induce a robust osteoclastogenic response.  r: d+ n$ |$ M% w- u
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To test this hypothesis, we treated cells with TNF- and M-CSF without the addition of RANK-L. Specifically, we examined osteoclastogenesis at varying doses of TNF- in bone marrow macrophages from wild-type and CD38-deficient mice. We found that the osteoclastogenic response of bone marrow macrophages to TNF- treatment alone was markedly enhanced in the absence of CD38 ( Fig. 1, A and B ). Despite the enhanced osteoclastogenesis, TNF- treatment alone at the doses tested remained unable to induce as profound an osteoclastogenic response as RANK-L ( Fig. 1 C ).4 u& n# K/ A7 S

Fig. 1. The absence of CD38 increases the propensity of bone marrow macrophages to form osteoclasts in response to TNF- alone. Ficoll-purified bone marrow cells were plated at 3 x 10 4 cells/well with 30 ng/ml murine M-CSF and varying concentrations of murine TNF-. After 5 days, the cells were stained for TRAP and a photograph taken at x 4 power on an inverted microscope ( A ). The number of TRAP( ) osteoclasts/well is shown ( B ). Values are means ± SE. C : note that RANK-L (60 ng/ml) leads to significantly greater osteoclast formation than the highest dose of TNF-.
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We next confirmed the enhanced osteoclastogenic potential of CD38-deficient cells in response to TNF- by measuring the upregulation of osteoclast differentiation markers by real-time PCR. We treated bone marrow macrophages with either RANK-L alone or TNF- alone and compared the upregulation of the osteoclast markers tartrate-resistant acid phosphosphatase ( TRAP ) and cathepsin K in wild-type cells to CD38-deficient ones. We found that the expression of both markers was enhanced in CD38-deficient cells in response to treatment with either RANK-L or TNF- ( Fig. 2 ). Moreover, we found that the expression of the proosteoclastogenic transcription factor c-fos was significantly greater in CD38-deficient cells than in wild-type cells ( Fig. 2 ). In contrast, the expression of the CD38 homolog CD157 was not differentially upregulated to compensate for the loss of CD38, as we have shown previously ( 10 ).5 D3 [9 C- i! d* ^& Y' r0 \3 ^6 Z
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These results suggest that CD38 upregulation on bone marrow macrophages in response to inflammatory stimuli negatively impacts their differentiation toward the osteoclast lineage. In contrast to the negative influence of CD38 on osteoclastogenesis, we postulated that CD38 upregulation by inflammatory stimuli might enhance the immune response in these cells. It is possible that CD38 may serve as a regulator of cell fate; that is, it may impede osteoclast formation but augment the immune response.$ V# A5 x' q5 ^5 w
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TNF- priming of the innate immune response requires CD38. Exposure of macrophages to inflammatory cytokines enhances the inflammatory reaction induced on a subsequent exposure to LPS ( 18 ). For example, infection with Cornyebacterium parvum leads to elevated levels of interferon- and TNF-, which in turn, enhance the inflammatory response to LPS exposure ( 30 ). These cytokine-priming actions are significant in that blocking the action of interferon- or TNF- with monoclonal antibodies prevents LPS-induced mortality and associated increases in inflammation ( 30 ).. V9 W% q. I$ U8 o; n, F$ p

While there have been no reported impairments in cytokine production in CD38-deficient mice ( 26 ), under noninflammatory conditions the majority of immune cells expressing CD38 are lymphocytes. Thus, while CD38 has been shown to play a role in adaptive immunity, with deficient B cell antibody responses and ineffective T cell priming ( 5, 25 ), its role in innate immunity is less clear.) n0 z+ a4 e" t2 t! G

Because TNF- dramatically increases CD38 expression in macrophages ( 11 ), we investigated whether TNF- -primed macrophages exhibit defects in their inflammatory response on a subsequent challenge with an inflammatory stimulus. In other words, defects caused by the lack of CD38 expression in macrophages may be most noticeable when CD38 is maximally expressed. Therefore, we treated wild-type and CD38-deficient mice with TNF- or control for 15 h and then challenged them with LPS ( Fig. 3 A ). It has been reported previously that CD38 crosslinking with a monoclonal antibody can enhance production of IL-12 in human macrophages ( 3 ). We examined plasma IL-12 levels and found that IL-12 was equally induced in naive wild-type and CD38-deficient mice in response to LPS ( Fig. 3 B ). However, wild-type mice primed with TNF- to increase CD38 expression had a greater peak amount of IL-12 compared with CD38-deficient cells primed with TNF- on LPS exposure ( Fig. 3 B ).6 F' B# @2 [% s+ f. N6 c+ e& e/ V9 i
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Fig. 3. TNF- priming of macrophages to upregulate CD38 enhances LPS-induced inflammation. A : schematic of how TNF- priming and LPS-stimulation were carried out either in vivo in mice or in vitro with murine bone marrow macrophages. B : TNF- priming of mice leads to a greater increase in peak IL-12 production in response to a challenge with LPS ( P

While the changes in plasma IL-12 levels were modest in magnitude, we confirmed that a TNF- -priming discrepancy existed in CD38-deficient mice by examining whether the same effect was observable in vitro using bone marrow macrophages. Similar to the in vivo situation, we found that the inflammatory response to LPS was similar in naive wild-type and CD38-deficient macrophages ( Fig. 3 C ). TNF- -primed wild-type macrophages, however, exhibited a greater increase in their production of cytokines such as IL-12 and IL-1; this augmentation was not observed in TNF- -primed CD38-deficient macrophages ( Fig. 3 C ).

CD38 upregulation leads to altered signal transduction and decreased HDAC acitivity. There are two possibilities through which CD38 upregulation could enhance the inflammatory response. One possibility is that CD38 affects the signaling pathways downstream of Toll-like receptors (TLRs; receptors for LPS). Alternatively, CD38 could modulate the proclivity of inflammatory gene induction, for example, by modulating cellular HDAC activity./ G" D2 |& g$ b  l3 Z* B- T

We first investigated the former possibility. That CD38-deficient cells treated with TNF- had higher levels of c-fos compared with wild-type cells ( Fig. 2 ) suggested that ERK1/2 activation might be differentially affected. We examined ERK1/2 and other cell signaling pathways in wild-type and CD38-deficient macrophages by Western blotting. LPS or RANK-L stimulation of naive (nonprimed) cells produced no discernable differences in signaling pathway activation ( Fig. 4 and Supplemental Fig. 1; all supplementary material can be found in the online version of this article). However, following TNF- priming there was a decrease in ERK1/2 phosphorylation in response to either LPS or RANK-L in wild-type cells but not in CD38-deficient cells [ Fig. 4; for LPS and RANK-L, on the right hand blot of ERK1/2 compare the 5-min wild-type (WT) to CD38-deficient (KO) bands]. This finding was highly significant in that ERK1/2 signaling decreases IL-12 production ( 6, 34 ) but is essential for osteoclastogenesis ( 22 ). Moreover, this finding is consistent with our previous results showing that wild-type cells had greater peak IL-12 levels ( Fig. 3 B ) and that CD38-deficient cells had a greater amount of c-fos mRNA expression following TNF- treatment ( Fig. 2 ).- y  a1 J7 A+ L, I& L0 ~6 i8 j. }

Fig. 4. TNF- -induced CD38 upregulation leads to downregulation of ERK1/2 phosphorylation. Bone marrow macrophages were treated with either control ( left set of blots) or TNF- for 12 h ( right set of blots) and then stimulated with LPS or RANK-L for either 5 or 15 min. Equal amounts of protein from cellular extracts were loaded for electrophoresis and then transferred for Western blotting of the following proteins: phosphorylated ERK1/2 [(p)-ERK1/2], phosphorylated S6 kinase [(p)-S6], phosphorylated JNK1/2 [(p)-JNK1/2], phosphorylated I B [(p)-I B ], phosphorylated p38 MAP kinase [(p)- p38], phosphorylated c-raf [(p)- c-raf], and pin1 as a loading control. There were no significant differences between wild-type (WT) and CD38-deficient (KO) macrophages in response to LPS or RANK-L in the naive state. However, after TNF- pretreatment, wild-type cells displayed a decrease in their ERK1/2 activation, while CD38 -/- cells displayed decreases in I B phosphorylation (NF- B activation).
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In addition to altered ERK1/2 activation, the blots showed that NF- B activation may also be differentially regulated by TNF- -induced CD38 upregulation. In the basal state, CD38-deficient cells displayed a robust phosphorylation of I B in response to LPS or RANK-L; however, the NF- B response of CD38-deficient cells after TNF- priming was mitigated, albeit modestly, as demonstrated by decreased I B phosphorylation ( Fig. 4 ).

Murine IL-12 expression is positively regulated by the NF- B protein c-Rel and negatively regulated by the AP-1 protein c-fos ( 28, 29 ). The relevance of the effect of CD38 on the ERK1/2 and NF- B pathways was next examined by analyzing c-Rel and c-fos recruitment to the IL-12p40 promoter. While there were no large differences in naive cells, we found that the recruitment of c-Rel to the IL-12 promoter in wild-type cells was markedly enhanced by TNF- pretreatment ( Fig. 5 A ). In contrast, c-Rel recruitment to the IL-12 promoter was not increased in CD38-deficient cells after TNF- priming, suggesting that CD38 is necessary for enhanced c-Rel recruitment. The recruitment of c-fos to the IL-12 promoter was not different in naive cells, but CD38-deficient cells displayed increased recruitment of c-fos with TNF- pretreatment ( Fig. 5 B ).

Fig. 5. TNF- -induced CD38 upregulation results in enhanced recruitment of c-Rel and c-fos to the IL-12 promoter and decreases cellular HDAC activity. Because wild-type cells expressed greater amounts of inflammatory cytokines in response to TNF- than CD38 -/- cells, we examined whether there was a differential recruitment of transcription factors to the IL-12 promoter or if there were differences in cellular HDAC activity. A and B : effect of TNF- priming on the recruitment of c-Rel and c-fos to the IL-12p40 promoter was examined by chromatin immunoprecipitation (ChIP). Bone marrow macrophages were pretreated with TNF- for 15 h and then treated with LPS or control for 1 h. After sonification and immunoprecipitation with anti-c-Rel or anti- c-fos antibodies, crosslinks were reversed and the amount of the IL-12p40 promoter was determined relative to the input fraction by quantitative PCR. The y -axis represents fold-change in DNA binding relative to the input plus standard deviations. C and D : bone marrow macrophages plated at 3 x 10 4 cells/well and treated with either TNF- or control for 12 h to determine the effects of TNF- pretreatment on cellular HDAC activity. The amount of cleavage of a fluorescent HDAC substrate was measured over time. To confirm specificity of cleavage, the HDAC inhibitor trichostatin A (10 nM) was added to some wells. TNF- treatment led to decreases in HDAC activity in wild-type cells ( D ). CD38 -/- cells, in contrast, reduced their HDAC activity only slightly in response to TNF-. The y -axis represents the percentage of wild-type control HDAC activity.

While the data thus far provided evidence that CD38 can affect the ERK1/2 and NF- B-signaling pathways to result in altered gene expression, we also investigated whether CD38 upregulation impacted HDAC activity. Differences in HDAC activity represent another divergence point between a proosteoclastogenic and proinflammatory response. Inhibition of HDAC activity increases cytokine-induced inflammatory gene induction ( 1, 33 ). In contrast, inhibition of HDAC activity inhibits osteoclastogenesis ( 2 ). To investigate whether TNF- -induced CD38 upregulation impacted HDAC activity, we treated bone marrow macrophages with TNF- or control for 15 h. We assayed HDAC activity by the addition of a cell-permeable HDAC substrate, the cleavage of which generated a fluorescent compound that could be measured in a fluorimeter over time.

We found that there were no differences in wild-type and CD38-deficient cells with the control solution ( Fig. 5 C ). However, after TNF- stimulation wild-type cells had a marked decrease in HDAC activity ( Fig. 5 D ). This finding is significant in that decreased HDAC activity would allow greater gene induction following a stimulus (such as we have observed for LPS in Fig. 3 C ). In contrast to wild-type cells, CD38-deficient cells did not display significant decreases in their HDAC activity following TNF- treatment ( Fig. 5 D ). These findings suggest that TNF- -induced decreases in HDAC activity occur in part through the upregulation of CD38. In addition, they suggest that CD38 exerts its negative impact on osteoclastogenesis and enhances inflammation through decreases in HDAC activity.
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DISCUSSION

What tells a pluripotent cell which path to follow? Bone marrow macrophages have a unique ability to differentiate into tissue macrophages, dendritic cells, or osteoclasts ( Fig. 6 ). Different cytokines are the primary determinants of their cell fate. However, how cytokines actualize such differentiation remains unclear; in fact, some cytokines seem crucial for multiple purposes. This is illustrated by the cytokine RANK-L. RANK-L is essential for osteoclastogenesis, but it is also essential for lymph node formation and acts to augment dendritic cell priming. How its expression in the bone marrow leads to osteoclast formation, while its expression in the lymphatic system leads to lymph node formation is unknown. We hypothesize that the environment in which the cytokines act strongly influences the outcome.) b' z% T" e! K# a3 N3 R+ C

Fig. 6. Differentiation potential of bone marrow monocytic precursors. Bone marrow monocytic precursors have the potential to differentiate into osteoclasts with exposure to RANK-L, into macrophages with exposure to M-CSF, or into dendritic cells with exposure to GM-CSF, IL-4, and TNF-. In addition to cytokine-regulated differentiation, monocytes and dendritic cells can become activated by exposure to inflammatory stimuli such as LPS or TNF-.+ J7 j: O3 o: H( J/ r: U

Here, we have shown that the response to a cytokine or pathogen can be influenced by the upregulation of the extracellular-facing enzyme CD38. It is likely that CD38 alters the local environment in which cytokines act and in doing so influences cell fate. We have focused on the effects exerted by TNF- -induced CD38 upregulation. It was suggested that the TNF- superfamily of cytokines evolved "to coordinate the social context" of immune responses enabling cells "to maximally respond to pathogens" ( 19 ). We have shown here that part of this coordination is realized through the upregulation of the ectoenzyme CD38, which acts to influence subsequent cytokine responses./ g, E( r2 ^+ s3 ]5 m
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We find that CD38 upregulation influences cell fate through multiple mechanisms. CD38 upregulation can decrease ERK1/2/ c-fos pathway responsiveness. While this decrease negatively influences osteoclastogenesis, it augments inflammatory cytokine (e.g., IL-12) production. Our results are consistent with findings that CD38 expression is negatively correlated with c-fos expression in human hematopoetic cells ( 23 ).! Y7 W2 i; }; H7 d5 ?$ b

Similar to c-fos, CD38-mediated increases in NF- B activation serve to augment inflammatory gene transcription. Previous investigations in B lymphocytes have demonstrated that CD38 ligation with monoclonal antibodies leads to the activation of c-Rel, p65, and p50 through pathways involving PKC and phosphatidylinositol 3-kinase ( 12 ). It remains to be investigated whether the augmentation of LPS- and RANK-L-induced NF- B activation shown here occurs through similar mechanisms. Moreover, it remains unclear whether the catalytic ability of CD38 is necessary for the observed changes in signaling pathways. If the catalytic ability is essential, then it is likely that ADP-ribose and nicotinamide, the products of CD38-catalyzed NAD breakdown, serve to alter the activation of signaling pathways. Nicotinamide has been shown previously to impact both ERK1/2 and NF- B activation ( 15, 16 ). The effects of ADP-ribose on NF- B and ERK1/2 activation are unknown and remain to be investigated.

Another mechanism through which CD38 upregulation can influence cell fate is by regulating cellular HDAC activity. We find that CD38 upregulation is necessary for TNF- -induced decreases in HDAC activity. By decreasing HDAC activity, CD38 likely enhances cytokine expression. This finding may in part explain the clinically observed connection between CD38 expression in chronic lymphocytic leukemia and enhanced production of cytokines, such as IL-4 ( 17 ). Moreover, decreases in HDAC activity oppose osteoclast formation ( 27 ), thus providing another means through which CD38 hinders osteoclastogenesis., l( r8 z) p$ ]5 Z$ g! I* Q% |

In summary, we have found that TNF- is capable of affecting the fate of macrophages by upregulating CD38. CD38, in turn, primes the cells to respond to further stimulation, such that signals induced by inflammatory stimuli are augmented, while those leading to osteoclast formation are inhibited. Priming plays an important role in disease and health; the result of uncontrolled priming is autoimmune-like diseases or multisystem organ failure ( 8 ). The work presented here on CD38 provides a unique paradigm for understanding how priming actualizes changes in the local environment, as well as how these changes modulate signal transduction and gene expression.) R9 _7 T: t' [. x
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This work was supported by National Institutes of Health Grants AG-14907, DK-70526, and AG-23176 (to M. Zaidi) and both an Endocrinology Training grant and MSTP Institutional grant (to J. Iqbal).
      【参考文献】
Adam E, Quivy V, Bex F, Chariot A, Collette Y, Vanhulle C, Schoonbroodt S, Goffin V, Nguyen TL, Gloire G, Carrard G, Friguet B, De Launoit Y, Burny A, Bours V, Piette J, Van Lint C. Potentiation of tumor necrosis factor-induced NF-kappa B activation by deacetylase inhibitors is associated with a delayed cytoplasmic reappearance of I kappa B alpha. Mol Cell Biol 23: 6200-6209, 2003.
( r" d0 Q0 P4 n6 J" O. I0 Y+ ?

$ z; Y% I5 a3 Y4 Z- k0 \; J9 V
Ashburner BP, Westerheide SD, Baldwin AS Jr. The p65 (RelA) subunit of NF-kappaB interacts with the histone deacetylase (HDAC) corepressors HDAC1 and HDAC2 to negatively regulate gene expression. Mol Cell Biol 21: 7065-7077, 2001., U  y( @4 I6 x$ ]

Ausiello CM, Urbani F, la Sala A, Funaro A, Malavasi F. CD38 ligation induces discrete cytokine mRNA expression in human cultured lymphocytes. Eur J Immunol 25: 1477-1480, 1995.

( d8 e0 m5 x4 Z2 z2 `& d

Banerjee D, Liou HC, Sen R. c-Rel-dependent priming of naive T cells by inflammatory cytokines. Immunity 23: 445-458, 2005.
( ]3 a9 ?, d% |  ^& Z% Z
( D+ O% ?8 \' }6 y
# q/ I8 M. z; R3 h
Cockayne DA, Muchamuel T, Grimaldi JC, Muller-Steffner H, Randall TD, Lund FE, Murray R, Schuber F, Howard MC. Mice deficient for the ecto-nicotinamide adenine dinucleotide glycohydrolase CD38 exhibit altered humoral immune responses. Blood 92: 1324-1333, 1998.
( c/ ?& Z. ?6 G: q, _/ Z" j! ]" I
3 f' L  p* I$ p
* @9 i+ S2 _. |3 @
Feng GJ, Goodridge HS, Harnett MM, Wei XQ, Nikolaev AV, Higson AP, Liew FY. Extracellular signal-related kinase (ERK) and p38 mitogen-activated protein (MAP) kinases differentially regulate the lipopolysaccharide-mediated induction of inducible nitric oxide synthase and IL-12 in macrophages: Leishmania phosphoglycans subvert macrophage IL-12 production by targeting ERK MAP kinase. J Immunol 163: 6403-6412, 1999.

; f+ O# h, H3 f) Z9 n5 P
Fulco M, Schiltz RL, Iezzi S, King MT, Zhao P, Kashiwaya Y, Hoffman E, Veech RL, Sartorelli V. Sir2 regulates skeletal muscle differentiation as a potential sensor of the redox state. Mol Cell 12: 51-62, 2003.* P/ m$ a7 ^: E! q
+ [" r7 n5 N' i0 s3 ~2 s$ F
+ Q3 ]7 Z4 s/ J$ B  C: |
% i4 q* c* H6 m$ t  ]! G# Y, r1 R
Goto M, Deriy LV, Chen YJ, Beno DW, Uhing MR, Jiyamapa-Serna VA, Kimura RE. TNF-alpha increases sensitivity to LPS in chronically catheterized rats. Am J Physiol Heart Circ Physiol 280: H2857-H2862, 2001.
2 v: {8 }4 B. `' x. G

" n/ K% ?7 u4 h! l* E9 I
Heiner I, Eisfeld J, Halaszovich CR, Wehage E, Jungling E, Zitt C, Luckhoff A. Expression profile of the transient receptor potential (TRP) family in neutrophil granulocytes: evidence for currents through long TRP channel 2 induced by ADP-ribose and NAD. Biochem J 371: 1045-1053, 2003." U- L4 D2 C$ q( |. w, Q5 ?2 }

Iqbal J, Kumar K, Sun L, Zaidi M. Selective upregulation of the ADP-ribosyl cyclases CD38 and CD157 by TNF but not by RANK-L reveals differences in downstream signaling. Am J Physiol Renal Physiol 291: F557-F566, 2006.

: I$ R' t$ z# s9 ]- Q3 G
Iqbal J, Zaidi M. TNF regulates cellular NAD  metabolism in primary macrophages. Biochem Biophys Res Commun 342: 1312-1318, 2006.
( q4 }/ n. K8 J* U. n; Y3 \( P
$ x& C! c/ W# e# ]

Kaku H, Horikawa K, Obata Y, Kato I, Okamoto H, Sakaguchi N, Gerondakis S, Takatsu K. NF-kappaB is required for CD38-mediated induction of C(gamma)1 germline transcripts in murine B lymphocytes. Int Immunol 14: 1055-1064, 2002.& r8 J5 S' Y. j0 \$ p2 Q

' s( ]6 _, o6 G9 D, T! j+ x
Kong YY, Feige U, Sarosi I, Bolon B, Tafuri A, Morony S, Capparelli C, Li J, Elliott R, McCabe S, Wong T, Campagnuolo G, Moran E, Bogoch ER, Van G, Nguyen LT, Ohashi PS, Lacey DL, Fish E, Boyle WJ, Penninger JM. Activated T cells regulate bone loss and joint destruction in adjuvant arthritis through osteoprotegerin ligand. Nature 402: 304-309, 1999.
6 `/ w+ j2 g. C6 D

' l9 u  J  q3 m+ [5 T
Kong YY, Yoshida H, Sarosi I, Tan HL, Timms E, Capparelli C, Morony S, Oliveira-dos-Santos AJ, Van G, Itie A, Khoo W, Wakeham A, Dunstan CR, Lacey DL, Mak TW, Boyle WJ, Penninger JM. OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature 397: 315-323, 1999.

- h0 }( J5 ?% V- w
) L: c5 c/ O# Z( J* @: [  \
Le Page C, Sanceau J, Drapier JC, Wietzerbin J. Inhibitors of ADP-ribosylation impair inducible nitric oxide synthase gene transcription through inhibition of NF kappa B activation. Biochem Biophys Res Commun 243: 451-457, 1998.8 s0 V8 ?" {: d' f4 I. e4 d& l
* {; g+ s# ^  R

3 \* j( e/ E5 d2 F5 B/ v" x
Le Page C, Wietzerbin J. Modulation of the activation of extracellular signal-regulated kinase (ERK) and the production of inflammatory mediators by ADP-ribosylation inhibitors. Biol Chem 384: 1509-1513, 2003.

Levesque MC, Chen Y, Beasley BE, O'Loughlin CW, Gockerman JP, Moore JO, Weinberg JB. Chronic lymphocytic leukemia cell CD38 expression and inducible nitric oxide synthase expression are associated with serum IL-4 levels. Leuk Res 30: 24-28, 2006.
; G$ n+ k8 y/ w" m

Lewkowicz P, Tchorzewski H, Dytnerska K, Banasik M, Lewkowicz N. Epidermal growth factor enhances TNF-alpha-induced priming of human neutrophils. Immunol Lett 96: 203-210, 2005.

Locksley RM, Killeen N, Lenardo MJ. The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell 104: 487-501, 2001.0 H) _3 i6 [9 `* T1 Q

Lovibond AC, Haque SJ, Chambers TJ, Fox SW. TGF-beta-induced SOCS3 expression augments TNF-alpha-induced osteoclast formation. Biochem Biophys Res Commun 309: 762-767, 2003.- Y) \0 C/ m  ^- u. J+ p2 U

" w$ I( m# ~2 g8 ?7 N3 n4 d! b
Morita K, Kitayama T, Kitayama S, Dohi T. Cyclic ADP-ribose requires FK506-binding protein to regulate intracellular Ca 2  dynamics and catecholamine release in acetylcholine-stimulated bovine adrenal chromaffin cells. J Pharmacol Sci 101: 40-51, 2006.
) z. Z- D0 l- G( V9 l3 a' t

0 K5 E; l9 S% X: j0 R
Nakamura H, Hirata A, Tsuji T, Yamamoto T. Role of osteoclast extracellular signal-regulated kinase (ERK) in cell survival and maintenance of cell polarity. J Bone Miner Res 18: 1198-1205, 2003.

# t1 u4 |* C( e% f7 i) w
Oh IH, Lau A, Eaves CJ. During ontogeny primitive (CD34 CD38 - ) hematopoietic cells show altered expression of a subset of genes associated with early cytokine and differentiation responses of their adult counterparts. Blood 96: 4160-4168, 2000.; l+ X$ @6 N3 q

8 `, \- c0 B+ }, P4 a7 n0 n
Partida-Sanchez S, Cockayne DA, Monard S, Jacobson EL, Oppenheimer N, Garvy B, Kusser K, Goodrich S, Howard M, Harmsen A, Randall TD, Lund FE. Cyclic ADP-ribose production by CD38 regulates intracellular calcium release, extracellular calcium influx and chemotaxis in neutrophils and is required for bacterial clearance in vivo. Nat Med 7: 1209-1216, 2001.6 v8 o: V/ U0 p  o6 M- x, ^

Partida-Sanchez S, Goodrich S, Kusser K, Oppenheimer N, Randall TD, Lund FE. Regulation of dendritic cell trafficking by the ADP-ribosyl cyclase CD38: impact on the development of humoral immunity. Immunity 20: 279-291, 2004., l  j" h6 Z" v$ t

- }& E/ n0 c# v2 ^# D; E2 V5 c0 N
5 Y. v3 W* K6 T. {
Partida-Sanchez S, Randall TD, Lund FE. Innate immunity is regulated by CD38, an ecto-enzyme with ADP-ribosyl cyclase activity. Microbes Infect 5: 49-58, 2003.' _( M. t- T6 Z* t- ^  T* Y# k8 k
1 P3 z# y! Q# y6 E: k! F

Rahman MM, Kukita A, Kukita T, Shobuike T, Nakamura T, Kohashi O. Two histone deacetylase inhibitors, trichostatin A and sodium butyrate, suppress differentiation into osteoclasts but not into macrophages. Blood 101: 3451-3459, 2003.

: ]2 [' S, @3 w. Y+ X* j

Ray N, Kuwahara M, Takada Y, Maruyama K, Kawaguchi T, Tsubone H, Ishikawa H, Matsuo K. c-Fos suppresses systemic inflammatory response to endotoxin. Int Immunol 18: 671-677, 2006.# M  n# @* a7 M
7 R6 u- z6 b6 u8 Q! v& h
) c& \- e# K( I8 D$ M
. s) L+ S( [# d" _
Sanjabi S, Hoffmann A, Liou HC, Baltimore D, Smale ST. Selective requirement for c-Rel during IL-12 P40 gene induction in macrophages. Proc Natl Acad Sci USA 97: 12705-12710, 2000.
2 C5 I3 n+ ~, F: Y* l
+ ~: b3 t  P8 k1 \3 a

Smith SR, Terminelli C, Denhardt G, Narula S, Thorbecke GJ. Administration of interleukin-10 at the time of priming protects Corynebacterium parvum -primed mice against LPS- and TNF-alpha-induced lethality. Cell Immunol 173: 207-214, 1996.0 U8 _: S5 `; [- n* @

" E) B, L/ h% R8 x& h4 U

Stager S, Kaye PM. CD8  T-cell priming regulated by cytokines of the innate immune system. Trends Mol Med 10: 366-371, 2004.2 B3 ]9 S$ h7 d5 _% ~' X
0 _6 @9 e5 \9 c

Sun L, Iqbal J, Dolgilevich S, Yuen T, Wu XB, Moonga BS, Adebanjo OA, Bevis PJ, Lund F, Huang CL, Blair HC, Abe E, Zaidi M. Disordered osteoclast formation and function in a CD38 (ADP-ribosyl cyclase)-deficient mouse establishes an essential role for CD38 in bone resorption. FASEB J 17: 369-375, 2003.! h% @+ s1 k6 l+ _

Suuronen T, Huuskonen J, Pihlaja R, Kyrylenko S, Salminen A. Regulation of microglial inflammatory response by histone deacetylase inhibitors. J Neurochem 87: 407-416, 2003.
- l) m- `: n. F  ]% o# s
0 I3 K6 h  P0 w% A+ d" |

Tomczak MF, Gadjeva M, Wang YY, Brown K, Maroulakou I, Tsichlis PN, Erdman SE, Fox JG, Horwitz BH. Defective activation of ERK in macrophages lacking the p50/p105 subunit of NF-kappaB is responsible for elevated expression of IL-12 p40 observed after challenge with Helicobacter hepaticus. J Immunol 176: 1244-1251, 2006.

  m# y" l' Q- D8 H

Yeung F, Hoberg JE, Ramsey CS, Keller MD, Jones DR, Frye RA, Mayo MW. Modulation of NF-kappaB-dependent transcription and cell survival by the SIRT1 deacetylase. EMBO J 23: 2369-2380, 2004.

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