Phospholipase Cγ2 Mediates RANKL-stimulated Lymph Node Organogenesis and Osteoclastogenesis*

Phospholipase Cγ2 (PLCγ2) is an important signaling effector of multiple receptors in the immune system. Here we show that PLCγ2-deficient mice displayed impaired lymph node organogenesis but normal splenic structure and Peyer's patches. Receptor activator of NF-κB ligand (RANKL) is a tumor necrosis factor family cytokine and is essential for lymph node organogenesis. Importantly, PLCγ2 deficiency severely impaired RANKL signaling, resulting in marked reduction of RANKL-induced activation of MAPKs, p38 and JNK, but not ERK. The lack of PLCγ2 markedly diminished RANKL-induced activation of NF-κB, AP-1, and NFATc1. Moreover, PLCγ2 deficiency impaired RANKL-mediated biological function, leading to failure of the PLCγ2-deficient bone marrow macrophage precursors to differentiate into osteoclasts after RANKL stimulation. Re-introduction of PLCγ2 but not PLCγ1 restores RANKL-mediated osteoclast differentiation of PLCγ2-deficient bone marrow-derived monocyte/macrophage. Taken together, PLCγ2 is essential for RANK signaling, and its deficiency leads to defective lymph node organogenesis and osteoclast differentiation.

PLC␥2 is a lipid enzyme, and activation of PLC␥2 hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol and inositol 1,4,5-trisphosphate (1,2). Both diacylglycerol and inositol 1,4,5-trisphosphate are important second signaling messengers for diverse cellular responses (1,2). Diacylglycerol activates protein kinase C (PKC), 3 whereas inositol 1,4,5-trisphosphate mediates the mobilization of Ca 2ϩ from internal stores, resulting in a transient intracellular Ca 2ϩ flux (1). Activated PKC, via a three component complex composed of CARMA1 (CARD, membrane-associated guanylate kinase, MAGUK, protein 1), Bcl10 (B-cell lymphoma protein 10), and MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1), leads to the activation of IB kinase (3)(4)(5). Activated IB kinase then phosphorylates a family of cytoplasmic inhibitory proteins IB, triggering its ubiquitination and proteolysis by the proteasome complex (5). Ultimately, the degradation of IB releases sequestration of transcription factors of the NF-B family in the cytoplasm, leading to its nuclear localization and activation of its target genes (6,7). Meanwhile, the elevated intracellular Ca 2ϩ binds to calmodulin, activating the serine/threonine phosphatase calcineurin. Activation of calcineurin leads to dephosphorylation of the transcription factor NFAT, resulting in its translocation from the cytoplasm to the nucleus and ultimate activation of its target genes (8). Moreover, the PLC␥/Ca 2ϩ /PKC pathway has been shown to participate in the activation of all types of MAP kinases (ERKs, JNKs, and p38 MAPKs) (9 -13) even though PKC-independent Grb2/ SOS/Raf1 pathway plays a primary role in the activation of MAPKs (10,14,15). Activated PKC can promote activation of ERK-1 and ERK-2 and is required for the maximum activation of p38 MAPK (12,13,16). In addition, calcium and PKC are involved in JNK activation (12,13). Ultimately, the activation of the three MAP kinase leads to the activation of transcription factors, including AP-1 (17)(18)(19).
PLC␥2 is primarily expressed in hematopoietic cell lineages (1). Targeted gene disruption studies have revealed a critical role of PLC␥2 in multiple receptor-mediated biological functions. PLC␥2 is essential for pre-B cell receptor (BCR)-and BCR-mediated B cell development and functions, and its deficiency affects early B cell development and severely impairs B cell maturation and responses to antigen challenges (20 -23). PLC␥2 is also a critical component of FcR␥ chain-containing collagen receptor signaling pathways, and deficiency of PLC␥2 results in platelet dysfunction and fetal hemorrhage (20). In addition, PLC␥2 participates in Fc⑀R signaling, and its deficiency impairs Fc⑀R-induced degranulation and cytokine secretion in mast cells (24). Last, PLC␥2 correlates with defective Fc␥R-mediated ADCC (antibody-dependent cell-mediated cytotoxicity) activity in NK cells (20) and is involved in signaling of the major activating receptor, NKG2D, of the NK cells (25,26), wherein PLC␥2 deficiency disrupts NKG2D-mediated NK cell maturation and function (27,28).
Receptor activator of NF-B ligand (RANKL) is a tumor necrosis factor family cytokine (29,30). RANKL is essential for early lymphocyte development and lymph node organogenesis (31,32). RANKL also mediates the final differentiation of bone marrow derived monocyte/macrophage precursors (BMMs) into osteoclasts (29,30,32), whereas macrophage-colony stimulating factor (M-CSF) controls the survival and proliferation of these precursors (33). RANKL deficiency impairs the early development of both T and B cells and blocks the formation of lymph nodes, resulting in immunodeficiency disease (32).
Here we demonstrate that PLC␥2 is activated upon RANKL stimulation. Importantly, deficiency of PLC␥2 severely impairs RANKL signaling and results in the lack of lymph node organogenesis in mice.

EXPERIMENTAL PROCEDURES
Mice-PLC␥2 Ϫ/Ϫ mice were as previously described (20). Bone morphology assessment was performed with age-and gender-matched PLC␥2 Ϫ/Ϫ and wild-type control littermates. All animal usage followed the guideline of the Institutional Animal Care and Use Committees at the University of Alabama at Birmingham and the Blood Research Institution at the Blood-Center of Wisconsin.
Deriving BMMs and RANKL-mediated Differentiation of BMMs in Vitro-In vitro osteoclastogenesis was performed as previously described (49,50). Briefly, bone marrow (BM) cells were isolated from long bones of 8 -12-week-old PLC␥2 Ϫ/Ϫ and wild-type control littermates. The cells were grown in complete ␣-MEM (Invitrogen) with murine M-CSF (10 ng/ml) for 72 h. Then the nonadherent BMMs were cultured with glutathione S-transferase-RANKL (100 ng/ml) and M-CSF (10 ng/ml). Cell differentiation was examined by TRAP staining according to the manufacturer's instruction (Sigma).
Retroviral Transduction of BMMs of PLC␥1 and PLC␥2-The retroviral transduction was as previously described (22). Briefly, rat PLC␥1 or rat PLC␥2 gene has been cloned into a vector with bicistronic retrovirus murine stem cell virus promoter-internal ribosome entry site (IRES)-GFP to generate GFP-IRES-PLC␥1 and GFP-IRES-PLC␥2 vectors. Conditioned media containing high titer, amphotropic retrovirus particles derived from 293T cells were filtered and used for transduction. Wild-type and PLC␥2 Ϫ/Ϫ BMMs were exposed to filtered conditioned media that contained PLC␥1 or PLC␥2 retrovirus for 2 days, and subsequently RANKL (100 ng/ml) and M-CSF (10 ng/ml) were added. Retrovirus with GFP alone served as the control. Media were changed every other day. The effects on osteoclastogenesis were determined by TRAP staining and normalized by measuring infection efficiency assessed by GFP expression.
Western Blot Analysis-Cells were lysed, and cell lysates were subjected to SDS-PAGE and Western blot analysis as previously described (52). For measuring activation of MAPKs, nonadherent BM cells were cultured in complete ␣-MEM with 10% fetal bovine serum and murine M-CSF (10 ng/ml) for 3 days. The cells were then starved in serum-free media for 6 h fol-lowed by stimulation with RANKL (100 ng/ml) for the indicated times. Subsequently, the cells were lysed and subjected to Western blot analysis with the indicated antibodies. For measuring up-regulation of NFATc1, nonadherent BM cells were cultured in complete ␣-MEM with 10% fetal bovine serum and murine M-CSF (10 ng/ml) for 2.5 days. The cells were then stimulated with RANKL (100 ng/ml) for 2.5 days and then subjected to Western blot analysis with the indicated antibodies.
Gel Mobility Shift Assays-For AP-1, nonadherent BM cells were cultured in complete ␣-MEM with 10% fetal bovine serum and murine M-CSF (10 ng/ml) for 3 days. The cells were cultured in serum-free medium with or without RANKL (100 ng/ml) or M-CSF (10 ng/ml) for 16 h. Nuclear extracts were prepared for gel mobility shift assays using 32 P-labeled probes containing AP-1 binding (purchased from Promega) or Oct-1 binding (purchased from Santa Cruz Biotechnology) sequences. For NF-B, nonadherent BM cells were cultured in complete ␣-MEM with 10% fetal bovine serum and murine M-CSF (10 ng/ml) for 3 days. The cells were starved in serumfree medium for 18 h and then stimulated with RANKL (100 ng/ml) for the indicated times. Nuclear extracts were prepared for gel mobility shift assays using 32 P-labeled probes containing NF-B (purchased from Promega) or Oct-1 binding sequences.
Bone Histomorphometry Analysis-Femurs were removed from the indicated mice and fixed in 10% formalin, decalcified in EDTA, and embedded in paraffin. Longitudinal sections (5 m thick) were stained with hematoxylin and eosin. Bone histomorphometry analysis was performed as previously described (53) with Bioquant image Analysis Software (R & M Biometrics). Various bone parameters, including bone thickness, osteoid surface, and osteoblast and osteoclast numbers were determined. Goldner's Trichrome staining was performed as previously described (53).
Statistical Analysis-Statistical analysis was performed as previously described (54). The differences between two groups were identified by Student's t tests. For multiple groups, one-way analysis of variance and Student-Newman-Keuls tests were used to identify differences. Significance was defined as p Ͻ 0.05.

RESULTS
PLC␥2 Deficiency Impairs Lymph Node Organogenesis-We examined the effect of PLC␥2 deficiency on lymph node organogenesis. Anatomical analysis of secondary lymphoid organs demonstrated that PLC␥2 Ϫ/Ϫ mice displayed dramatically impaired mesenteric, cervical, mandibular, inguinal, axillary, para-aortic, and popliteal lymph nodes compared with wildtype mice ( Fig. 1A and data not shown). Thus, PLC␥2 deficiency severely impairs lymph node organogenesis, similar to either the lack of lymphotoxin pathway or RANKL pathway.
Previous studies have linked the formation of lymph node with that of Peyer's patch and splenic architecture in the lymphotoxin pathway but not in the RANKL pathway. Lymphotoxin-deficient mice lack lymph nodes and also display defects in the formation of Peyer's patch and exhibit disorganized splenic architecture (55)(56)(57)(58). Interestingly, despite the severe defects of all lymph nodes in PLC␥2 Ϫ/Ϫ mice, the mutant mice had normal Peyer's patches compared with wild-type mice (Fig.   1B). Moreover, PLC␥2 Ϫ/Ϫ mice exhibited intact splenic architecture, including normal distribution of red and white pulp and normal primary follicle structure, including T-and B-cell areas and marginal zones ( Fig. 1C and data not shown). Taken together, these data demonstrate that PLC␥2 is essential for lymph node but not Peyer's patch or splenic architecture formation, which is consistent with the role of RANKL.
PLC␥2 Is Activated by RANKL in BMMs-RANKL has been shown to play an essential role in lymph node organogenesis (32). The critical role of PLC␥2 in lymph node organogenesis prompted us to determine the role of PLC␥2 in RANKL-mediated signaling and biological functions. BMMs are derived from BM cells upon M-CSF treatment (33). RANK, the receptor for RANKL on BMMs, mediates their final differentiation (29,30,32). To determine whether PLC␥2 is involved in RANKL signaling, we first examined its expression in BMMs by Western blot analysis. Wild-type BMMs expressed high levels of PLC␥2 proteins, whereas PLC␥2 Ϫ/Ϫ BMMs lacked the protein ( Fig.  2A). In addition, wild-type BMMs also expressed PLC␥1, the other family member of PLC␥, and PLC␥2 deficiency had no effect on the expression of PLC␥1 in the cells (Fig. 2A).
Next, we examined whether PLC␥2 plays a role in RANKL signaling. BMMs derived from wild-type mice were stimulated with RANKL, and activation of PLC␥2 was measured by its tyrosine phosphorylation, which is known to correlate with its lipase activity (59 -62). RANKL induced activation of PLC␥2 by as early as 2 min (Fig. 2B). Taken together, these data demon- strate that PLC␥2 is expressed in BMMs and is activated by RANKL.
PLC␥2 Deficiency Severely Impairs RANKL Signaling-To further determine whether PLC␥2 plays an important role in RANKL signaling, we examined the effect of PLC␥2 deficiency on RANKL signaling. BMMs were derived from wild-type and PLC␥2 Ϫ/Ϫ mice. Both BMMs expressed comparable levels of myeloid cell lineage markers CD14 and Mac-1 (Fig. 2C). PLC␥2 Ϫ/Ϫ BMMs also displayed normal levels of M-CSF receptor c-Fms relative to wild-type cells (Fig. 2C). Importantly, PLC␥2 Ϫ/Ϫ BMMs exhibited normal levels of RANK relative to wild-type cells (Fig. 2C). Therefore, lack of PLC␥2 does not affect M-CSF-mediated BMM development or reduce RANK expression on these precursors.
PLC␥2 Deficiency Severely Impairs RANKL-mediated Osteoclast Formation-Last, we examined the effect of PLC␥2 deficiency on RANKL-mediated biological function. In the presence of M-CSF, RANKL induced wild-type BMMs to differentiate into giant multinucleated and TRAP-positive osteoclasts, whereas PLC␥2 Ϫ/Ϫ BMMs failed to become multinucleated osteoclasts, although some of them became TRAP-positive (Fig. 4A). Moreover, semiquantitative RT-PCR demonstrated that RANKL-induced expression of TRAP and other osteoclast-associated genes, such as Cath K and CTR, was markedly impaired in PLC␥2 Ϫ/Ϫ compared with wild-type BMMs (Fig. 4B). Of note, both wild-type and PLC␥2 Ϫ/Ϫ BMMs expressed comparable levels of RANK before and after RANKL stimulation (Fig. 4B), consistent with the results from fluorescence-activated cell sorter analysis shown in Fig. 2C. Furthermore, the marked impairment of RANKL-induced expression of TRAP, Cath K, and CTR in PLC␥2 Ϫ/Ϫ cells was confirmed by quantitative real-time RT-PCR analyses (Fig. 4C). Taken together, these data demonstrate that PLC␥2 deficiency impairs RANKL-mediated biological functions, e.g. expression of osteoclastogenesis-associated genes, TRAP, Cath K, and CTR.
Furthermore, we examined the effect of PLC␥2 deficiency on RANKL-mediated osteoclast development in vivo. We evaluated the bone morphology of PLC␥2-deficient mice. The bone mineral density of femurs derived from wild-type and PLC␥2 Ϫ/Ϫ mice was analyzed using dual-energy x-ray absorptiometry (67). Femurs from PLC␥2 Ϫ/Ϫ mice exhibited an increased bone mineral density compared with those from wild-type mice (Fig. 4D). Histological examination of the femurs using Goldner's Trichrome staining also revealed a marked increase in bone density in PLC␥2 Ϫ/Ϫ relative to wild-type littermate control mice (Fig. 4E, upper). Microcomputed tomography evaluation of the femurs further demonstrated that bone density in PLC␥2 Ϫ/Ϫ mice was markedly increased compared with wild-type littermate controls (Fig. 4E, lower). Moreover, quantitative measurements of the ratio of trabecular bone volume (BV) to total bone volume (TV), BV/TV, an indicator of bone mass, showed a 2-fold increase in PLC␥2 Ϫ/Ϫ relative to wild-type mice, and importantly, the increased bone volume in PLC␥2 Ϫ/Ϫ mice was associated with a decreased number of osteoclasts (N.Oc/Bs) and decreased osteoclast surface (OcS/Bs) (data not shown). These data demonstrate that PLC␥2 is important for RANKL-mediated biological function in vivo.

DISCUSSION
RANKL is an important cytokine for early lymphocyte development and lymph node organogenesis (29 -32). The Ca 2ϩ -dependent activation of NFAT and the PKC-dependent activation of NF-B are indispensable for RANKL-mediated biological BMMs were starved in serum-free medium for 18 h and then stimulated with RANKL (100 ng/ml) for 0, 15, or 30 min. Nuclear extracts were subjected to gel mobility shift assays using 32 P-labeled probes containing NF-B or Oct-1 binding sequences. F, RANKL-mediated activation of AP-1 in PLC␥2-deficient BMMs. BMMs were cultured in serum-free medium with or without RANKL or M-CSF for 16 h. Nuclear extracts were subjected to gel mobility shift assays using 32 P-labeled probes containing AP-1-binding or Oct-1binding sequences.
functions (42,46). RANKL is a tumor necrosis factor family cytokine (29,30), and thus, the mechanism by which RANKL activates the Ca 2ϩ -dependent pathway has been a puzzle (42). The recruitment of the immunoreceptor tyrosine-based activation motif-containing adaptor proteins, DAP12 and FcR␥, to the RANK receptor complex has been shown to play a role in RANKL-induced Ca 2ϩ -flux (34 -36). PLC␥2 is involved in signaling of the FcR␥-chain-containing collagen receptor (20) and the DAP12-associated NKG2D receptor (27,28). Here we report that PLC␥2 plays a critical role in RANKL-induced activation of p38, JNK, AP-1, NF-B, and NFAT and in RANKL-mediated lymph node organogenesis and osteoclastogenesis. It is highly possible that immunoreceptor tyrosine-based activation motif-containing DAP12 and FcR␥ may play a role in RANKL-mediated activation of PLC␥2. Consistent with this notion, deficiency of DAP12 or FcR␥ disrupts RANKL-induced signaling and RANKL-mediated biological functions such as osteoclastogenesis, a defect very similar to that observed in PLC␥2 Ϫ/Ϫ mice (34 -36, 70).
Although PLC␥2 deficiency severely impairs RANKL signaling and RANKL-mediated cell differentiation, PLC␥2 Ϫ/Ϫ mice exhibit markedly reduced osteoclasts that appear normal BMMs derived from wild-type and PLC␥2-deficient mice were cultured with RANKL and M-CSF for 6 days. The cells were stained with TRAP staining and visualized by light microscopy. B, RT-PCR analyses of RANKL-induced expression of differentiation associated genes in PLC␥2-deficient BMMs. RANKL-stimulated BMMs from A were subjected to RT-PCR analyses of expression of the indicated genes. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. C, real-time RT-PCR analyses of RANKL-induced expression of differentiation associated genes in PLC␥2-deficient BMMs. RANKL-stimulated BMMs from A were subjected to quantitative real-time RT-PCR analyses of expression of the indicated genes. The expression data of each gene were normalized to the levels of glyceraldehyde-3-phosphate dehydrogenase expression. D, an increased bone mineral density of femurs derived from PLC␥2-deficient mice. Femurs derived from wild-type (PLC␥2 ϩ/ϩ ) and PLC␥2-deficient (PLC␥2 Ϫ/Ϫ ) mice were subjected to dual-energy x-ray absorptiometry analysis of bone mineral density. E, histology of the femurs from PLC␥2-deficient mice by Goldner's Trichrome and microcomputed tomography evaluation. Femurs derived from wild-type and PLC␥2deficient mice were subjected to Goldner's Trichrome (upper) and microcomputed tomography evaluation (lower). The figures shown A, D, and E are representative of six pairs of age-and gender-matched wild-type and PLC␥2-deficient littermates. The figures shown in B and C are representative of three independent analyses.
grossly. It is plausible that PLC␥1 isoform, which is expressed in BMMs, may compensate PLC␥2 deficiency in RANKL-mediated biological function in vivo. PLC␥1 and PLC␥2 have been shown to have similar functions in pre-B cell receptor-mediated development of early B cells (22). Equally possible, other PLC␥2-independent signaling pathway may be able to support differentiation of PLC␥2 Ϫ/Ϫ osteoclasts in vivo. PLC␥1 deficiency results in early embryonic lethality (71), and thus, conditional deletion of PLC␥1 in BMMs will help to clarify this issue. Because both PLC␥1 and PLC␥2 produce same secondary messages diacylglycerol and inositol 1,4,5-trisphosphate upon activation, it is a bit surprising to observe that overexpression of PLC␥1 fails to restore the ability of PLC␥2 Ϫ/Ϫ BMMs to differentiate to osteoclasts in vitro. A previous study has demonstrated that overexpression of PLC␥1 also fails to restore B cell receptor-mediated functions in PLC␥2 Ϫ/Ϫ late B cells (22). PLC␥2 is very likely to have a unique role in RANKL-mediated biological function, including lympho-node organogenesis, even if PLC␥1 might participate in RANKL signaling.
PLC␥2 activates multiple pathways, including PKC-dependent, Ca 2ϩ -dependent, and MAPK-dependent pathways (1,2). Deficiency of NF-B, whose activation is PKC-dependent, impairs RANKL-mediated function (46). Lack of NFATc1, a Ca 2ϩ pathway-dependent transcription, also interferes with RANKL-mediated biological function (42). However, the expression level of NFATc1 is very low in naïve BMMs. RANKL stimulation results in the activation of NFATc1, which turns on its own gene and results in the elevation of its own protein expression (42). In contrast, the expression level of NF-B is high in naïve BMMs, and NF-B is quickly activated upon RANKL stimulation. In addition, the JNK/AP-1 pathway is essential for RANKL-mediated osteoclast development (47). The multiple pathways, which are regulated by PLC␥2, are required for RANKL-mediated biological functions. Cross-talk among these signaling pathways in regulation RANKL-mediated biological functions likely exists. Indeed, studies have shown that NFATc1 cooperates with the AP-1 component c-Fos to promote expression of osteoclast-specific genes TRAP, cathepsin K, and CTR (42,72,73).
Organogenesis of lymph node or other secondary lymphoid organs, such as spleen and Peyer's patch, is highly regulated by chemokines and cytokines (74,75). For instance, the chemokine CXCL13 is required for secondary lymphoid organ development and deficiency of CXCL13, or its receptor CXCR5 severely impairs development of lymph node and Peyer's patch and organization of splenic microarchitecture (76,77). The tumor necrosis factor family of cytokines, such as lymphotoxin (LT)␣ and LT␤, are also essential for secondary lymphoid organ development, and mice deficient in LT␣ or LT␤ lack lymph node and Peyer's patch and have disorganized splenic microarchitecture (55,56,78,79). Activation of transcription factor NF-B seems to be a common pathway that controls organogenesis of lymph nodes, spleens, and Peyer's patches. Mice deficient in IB kinase ␣ or NF-B-inducing kinase (NIK), important kinases in NF-B activation, have severe defects in lymph nodes and Peyer's patches (80,81). However, organogenesis of lymph node also has its distinct requirement. For example, mice deficient in RANKL or and its receptor RANK lack lymph nodes but have normal Peyer's patches and organization of splenic microarchitecture (32,82). Interestingly, our current study has found that PLC␥2 plays an essential role in RANKL/ RANK signaling, and its deficiency specifically blocks organogenesis of lymph node but not Peyer's patch or spleen. Of note, LT-induced up-regulation of VCAM (vascular cell adhesion molecule), a NF-B-dependent event (83), is normal in the absence of PLC␥2 (data not shown). Thus, RANKL/RANK mediates lymph node organogenesis through signaling molecule PLC␥2, whereas LT␣/LT␤ does not depend on PLC␥2 in regulating organogenesis of peripheral lymphoid organs.