QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Supplemental Research Data Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Grabiner, B. C. Articles by Lin, X. Search for Related Content PubMed PubMed Citation Articles by Grabiner, B. C. Articles by Lin, X. Social Bookmarking                   What's this?

CARMA3 deficiency abrogates G protein-coupled receptor-induced NF-B activation

¹ Department of Molecular and Cellular Oncology, The University of Texas, M.D. Anderson Cancer Center, Houston, Texas 77030, USA; ² Department of Experimental Therapeutics, The University of Texas, M.D. Anderson Cancer Center, Houston, Texas 77030, USA; ³ Department of Immunology, The University of Texas, M.D. Anderson Cancer Center, Houston, Texas 77030, USA; ⁴ The CBR Institute for Biomedical Research, Harvard Medical School, Boston, Massachusetts 02115, USA

	Abstract

Top Abstract Results Discussion Materials and methods Acknowledgments References

 
G protein-coupled receptors (GPCRs) play pivotal roles in regulating various cellular functions. Although many GPCRs induce NF-B activation, the molecular mechanism of GPCR-induced NF-B activation remains largely unknown. CARMA3 (CARD and MAGUK domain-containing protein 3) is a scaffold molecule with unknown biological functions. By generating CARMA3 knockout mice using the gene targeting approach, here we show CARMA3 is required for GPCR-induced NF-B activation. Mechanistically, we found that CARMA3 deficiency impairs GPCR-induced IB kinase (IKK) activation, although it does not affect GPCR-induced IKK/ phosphorylation, indicating that inducible phosphorylation of IKK/ alone is not sufficient to induce its kinase activity. We also found that CARMA3 is physically associated with NEMO/IKK, and induces polyubiquitination of an unknown protein(s) that associates with NEMO, likely by linking NEMO to TRAF6. Consistently, we found TRAF6 deficiency also abrogates GPCR-induced NF-B activation. Together, our results provide the genetic evidence that CARMA3 is required for GPCR-induced NF-B activation.

[Keywords: NF-B; GPCR; CARMA3; neural tube]

Received October 13, 2006; revised version accepted February 20, 2007.

The NF-B family of transcription factors plays critical roles in controlling expression of survival factors, cytokines, and proinflammatory molecules (Ghosh et al. 1998). Stimulation of various cell surface receptors, including receptors for proinflammatory cytokines such as TNF and IL-1, Toll-like receptors (TLRs), antigen receptors, and GPCRs, activates NF-B by initiating distinct signaling pathways that eventually converge on the IB kinase (IKK) complex (Hayden and Ghosh 2004). The activated IKK phosphorylates the inhibitory molecule, IB, triggering the rapid ubiquitination and proteolysis of IBs. This unmasks the nuclear localization sequence of NF-B, resulting in its rapid translocation from the cytoplasm into nucleus, where it regulates the transcription of its target genes (Karin and Ben-Neriah 2000).

The IKK complex contains three subunits: two catalytic subunits, IKK and IKK, and an essential regulatory subunit, IKK/NEMO (Karin and Ben-Neriah 2000). Activation of the IKK complex is associated with phosphorylation of Ser177 and Ser181 in the activation loop of IKK and Ser176 and Ser180 in IKK. Genetic studies show that NEMO is required for the activation of NF-B by facilitating the formation of the high-molecular-weight IKK complex (Yamaoka et al. 1998). More recent studies have shown that Lys63 (K63)-linked polyubiquitination of NEMO is also essential for activating the IKK complex (Tang et al. 2003; Zhou et al. 2004). However, it is unclear whether IKK/ phosphorylation and K63-linked polyubiquitination of NEMO are regulated in a linear cascade or two parallel pathways.

CARMA3, Caspase recruitment domain (CARD)-associated and membrane-associated guanylate kinase domain (MAGUK)-containing protein 3 (Gaide et al. 2001; McAllister-Lucas et al. 2001; L. Wang et al. 2001), is a member of the CARMA family that contains three proteins, CARMA1, CARMA2, and CARMA3. These three proteins share similar structural motifs, with an N-terminal CARD domain, followed by a coiled-coil (CC) domain, a PDZ domain, a SH3 domain, and a C-terminal guanylate kinase-like (GUK) domain (Gaide et al. 2001; McAllister-Lucas et al. 2001; L. Wang et al. 2001). However, they have distinct expression patterns with CARMA1 (CARD11) expressed in hematopoietic cells, CARMA2 (CARD14/Bimp2) in the placenta, and CARMA3 (CARD10/Bimp1) in all nonhematopoietic cells (Bertin et al. 2001; Gaide et al. 2001; McAllister-Lucas et al. 2001; L. Wang et al. 2001). Recent studies have revealed a key role for CARMA1 in antigen receptor-induced NF-B activation (Gaide et al. 2002; Pomerantz et al. 2002; Wang et al. 2002; Egawa et al. 2003; Hara et al. 2003; Jun et al. 2003; Newton and Dixit 2003). In contrast, although the overexpression of CARMA2 and CARMA3 in HEK293 cells also induces NF-B activation (Gaide et al. 2001; McAllister-Lucas et al. 2001; L. Wang et al. 2001), the signaling pathway mediated by these proteins remains completely unknown.

To reveal the signaling pathways mediated by CARMA3, we used a gene targeting approach to generate CARMA3 knockout mice. Using CARMA3-deficient cells, we demonstrate that CARMA3 is required for GPCR-induced NF-B activation. This defect is specific because other stimuli such as TNF, lipopolysaccharide (LPS), and extracellular matrix proteins can activate NF-B in CARMA3-deficient cells. Together, our results reveal a new GPCR-induced signaling pathway that leads to NF-B activation.

	Results

Top Abstract Results Discussion Materials and methods Acknowledgments References

 

Generation of CARMA3-deficient mice

To investigate the biological function of CARMA3, we constructed a gene targeting vector that replaced the exon 3, which encodes a large part of the CARD domain, of the mouse Carma3 gene with a PGK-neo cassette (Fig. 1A). The targeting vector was electroporated into mouse embryonic stem (ES) cells. Two ES cell lines with the targeted allele were obtained after homologous recombination (data not shown) and used to generate chimeric mice. The targeted allele successfully underwent germline transmission. We were able to confirm that the Carma3 gene was disrupted in the targeted mice using genomic PCR, RT–PCR, and Western blot analysis (Fig. 1B–D). Of note, although the PGK-neo cassette (1 kb) insertion did not abolish Carma3 mRNA expression (Fig. 1C, lanes 2,3), it disrupted the protein expression of Carma3 (Fig. 1D), since the PGK-neo cassette was inserted into the exon 3 Carma3 gene in the reverse orientation (Fig. 1A). To further confirm that the detected Carma3 mRNA in CARMA3 mutant mice (Fig. 1C, lanes 2,3) could not express a functional CARMA3 protein, we amplified the mutant Carma3 cDNA by RT–PCR using mRNA from Carma3^–/– cells. Sequencing analysis indicates that the mutated Carma3 mRNA contains multiple stop codons in the insertion region of the PGK-neo cassette, thereby completely disrupting the CARMA3 coding sequence (data not shown) and resulting in a lack of expression of CARMA3 proteins (Fig. 1D).

View larger version (38K): [in this window] [in a new window]   Figure 1. The generation of CARMA3-deficient mice. (A) The targeting vector containing the PGK-Neor gene was used to replace Exon 3 of the Carma3 gene. Exon 3 of Carma3 encodes the majority of the CARD domain. Solid arrows indicate the sites of primers used for genotyping the Carma3 gene, whereas arrows indicate the sites of primers used for RT–PCR of CARMA3 mRNA. (B) Genomic PCR indicates the DNA rearrangement. (C) RT–PCR indicates the insertion of the Neo cassette into the Carma3 mRNA transcript in CARMA3-deficient mice. (D) Liver cell lysates from Carma3+/+ or Carma3–/– mice were subjected to SDS-PAGE and Western blot analysis using an anti-C-terminal CARMA3 antibody. (E) Carma3+/– and Carma3–/– E13.5 embryos exhibit the anencephaly phenotype of the neural tube closure defect. (F) Head sections of Carma3+/– and Carma3–/– E13.5 embryos were stained with H&E.

CARMA3 is required for LPA-induced NF-B activation

We next investigated in which signaling pathway CARMA3 is involved. Our earlier studies showed that CARMA3 could effectively rescue the defect of T-cell receptor (TCR)-induced NF-B activation in CARMA1-deficient T cells (Matsumoto et al. 2005), suggesting that CARMA3 and CARMA1 have similar upstream components, but in different signaling pathways. Since PKC functions upstream of CARMA1 in the signaling pathway of antigen receptors in lymphocytes (Matsumoto et al. 2005; Sommer et al. 2005), we postulated that PKC also functions upstream of CARMA3 in an unknown signaling pathway(s) in nonhematopoietic cells. Earlier studies have suggested that PKC is involved in NF-B activation induced by GPCRs (Shahrestanifar et al. 1999; Cummings et al. 2004), integrins (Juliano 2002), and receptor tyrosine kinases (RTKs) (Biswas et al. 2000). Therefore, we hypothesized that CARMA3 might function downstream from PKC in signaling pathways induced by GPCRs, integrins, or RTKs.

To test this hypothesis, we obtained mouse embryonic fibroblasts (MEF) from day 13.5 embryos of Carma3^+/+, Carma3^+/–, and Carma3^–/– mice. These MEF cells were stimulated with or without LPA. LPA is a potent bioactive phospholipid that induces cell survival and proliferation through its GPCRs (Moolenaar et al. 1997). Stimulation of cells with LPA could effectively induce NF-B activation in the presence of CARMA3. However, LPA-induced NF-B activation was abolished in Carma3^–/– cells, whereas TNF could effectively induce NF-B activation in the same cells (Fig. 2A, top panel). In addition, consistent with previous observations that PKC is involved in GPCR-induced NF-B activation, treatment of Carma3^–/– MEF cells with the pharmacological PKC agonists, phorbol-12-myristate-13-acetate (PMA) plus ionomycin (Iono), failed to induce NF-B activation (Fig. 2A, top panel). In contrast, LPA or PMA plus Iono-induced AP-1 activation was not significantly affected in the absence of CARMA3 (Fig. 2A, middle panel). Since the NF-B complex induced by LPA and PMA plus Iono mainly contained p65 and p50 (Fig. 2B), this result indicates that LPA-induced NF-B activation is through the classical NF-B pathway. To further confirm the requirement of CARMA3 for GPCR-induced NF-B activation, we reconstituted CARMA3-deficient MEF cells with an expression plasmid encoding HA-tagged CARMA3 (Supplementary Fig. 2), and found that NF-B activation (Fig. 2C) and IB phosphorylation (Supplementary Fig. 2) induced by LPA or PMA plus Iono were fully restored. Together, these results demonstrate that CARMA3 is required for GPCR-induced NF-B activation.

View larger version (65K): [in this window] [in a new window]

Since earlier studies suggest that PKC is also involved in integrin-induced NF-B activation (Juliano 2002), we hypothesized that integrin-induced NF-B also depends on CARMA3. However, we found that osteopontin (OPN) and RGD peptides, which can activate the _v3 integrin molecules, effectively induced NF-B activation in wild-type as well as CARMA3-deficient cells (Fig. 2E). In addition, although RTKs have been reported to activate NF-B through a PKC-dependent mechanism (Biswas et al. 2000), we were unable to detect NF-B activation in both wild-type and CARMA3-deficient MEF cells with FGF, PDGF, and IGF (data not shown). Therefore, we were unable to determine whether RTK-induced NF-B is dependent on CARMA3. Furthermore, we found that TLR-induced NF-B activation was not defective in CARMA3-deficient cells (data not shown). Together, our results indicate that CARMA3 is selectively involved in GPCR-induced NF-B activation.

To investigate the molecular mechanism by which LPA induces NF-B activation, we examined whether IB phosphorylation was defective in CARMA3-deficient cells. Consistent with the nuclear translocation of NF-B, stimulation with LPA could induce IB phosphorylation in Carma3^+/+, but not Carma3^–/–, cells, whereas TNF effectively induced IB phosphorylation and degradation in both Carma3^+/+ and Carma3^–/– cells (Fig. 3A, top panel), indicating that CARMA3 is specifically required for GPCR-induced IKK activation. Of note, LPA-induced IB phosphorylation was significantly weaker than that induced by TNF (Fig. 3A, top panel), and only a small portion of IB was degraded in wild-type cells (Fig. 3A, middle panel). To further demonstrate that the signal-induced IB degradation is required for LPA-induced NF-B, we pretreated cells with the proteasome inhibitor MG132 to block IB degradation. We found that NF-B activation induced by LPA, PMA plus Iono, or TNF was completely inhibited by the treatment of MG132 (Fig. 3B), indicating that the signal-induced IB degradation is required for LPA-induced NF-B activation.

View larger version (46K): [in this window] [in a new window]   Figure 3. LPA-induced IB phosphorylation is defective in CARMA3-deficient cells. (A) Carma3+/+ and Carma3–/– MEF cells were stimulated with or without LPA (10 µM) or TNF (10 ng/mL) for indicated time points. Cell lysates were then subjected to SDS-PAGE and Western blot analysis using indicated antibodies. (B) Carma3+/+ and Carma3–/– MEF cells were pretreated with or without MG132 for 15 min, then stimulated with or without LPA (10 µM) or TNF (10 ng/mL) for 1 h. Nuclear extracts were prepared from these cells, and subjected to EMSA using 32P-labeled NF-B or OCT-1 probe. (C) Carma3+/– and Carma3–/– MEF cells were stimulated with or without LPA (10 µM) for indicated time points. Cell lysates were then subjected to SDS-PAGE and Western blot analysis using indicated antibodies.

CARMA3-regulated IKK complex activation is independent on IKK/ phosphorylation

To investigate the molecular mechanism that results in the defect of IB phosphorylation in CARMA3-deficient cells, we examined IKK activation through an in vitro kinase assay following the stimulation of wild-type and CARMA3-deficient cells with LPA, PMA plus Iono, or TNF. We found that IKK activation was completely defective in response to the stimulation of LPA and PMA plus Iono in CARMA3-deficient cells, whereas TNF-induced IKK activation was comparable in both cell types (Fig. 4A). Consistent with this result, IB phosphorylation induced by LPA and PMA plus Iono was defective in CARMA3-deficient cells, but not in wild-type cells, whereas TNF effectively induced IB phosphorylation in both wild-type and CARMA3-deficient cells (Fig. 4B, top panel). Since IKK-mediated phosphorylation of p65 at Ser 536 has been linked to NF-B activation (Sakurai et al. 1999), we also examined the phosphorylation of p65 using antibodies against the phospho-Ser536 of p65. We found that the inducible phosphorylation on Ser536 was defective following the stimulation of LPA or PMA plus Iono in CARMA3-deficient cells (Fig. 4B, middle panel). Consistent with the above results, we surprisingly found that IKK phosphorylation, induced by the stimulation of LPA or PMA plus Iono, was not defective in CARMA3-deficient cells (Fig. 4B, bottom panel). Together, these results indicate that IKK activation is dependent on CARMA3 in the signaling pathways induced by GPCRs. However, although a CARMA3-independent signaling event can lead to IKK phosphorylation in GPCR signaling pathways, the inducible phosphorylation of IKK is insufficient to activate the IKK complex. This suggests that the full IKK complex activation is dependent on other CARMA3-associated signaling events.

View larger version (49K): [in this window] [in a new window]   Figure 4. CARMA3 regulates IKK kinase activity by inducing NEMO polyubiquitination but not IKK phosphorylation. (A) Carma3+/+ and Carma3–/– MEF cells were stimulated with or without LPA (10 µM), PMA (40 ng/mL) plus Iono (100 ng/mL) (P/I), or TNF (10 ng/mL) for indicated time points. IKK complex was immunoprecipitated using anti-IKK/ antibody-conjugated beads. The immunoprecipitated complex was subjected to an in vitro kinase assay using GST-IB as substrates. (B) Total cell lysates were subjected to SDS-PAGE and Western blot analysis using indicated antibodies. (C) HEK293 cells (5 x 105/well) were transfected with the plasmid encoding Flag-tagged CARMA3. Twenty hours later, cell lysates were prepared from the transfected cells and subjected to immunoprecipitation using anti-TAK1- or anti-NEMO-conjugated beads. The immunoprecipitated complexes and cell lysates were subjected to Western blot analysis using indicated antibodies. (D) The lysates from C were also immunoprecipitated using anti-NEMO- or control IgG-conjugated beads. The obtained immunocomplexes were washed with lysis buffer (containing 1% NP-40 and 250 mM NaCl) four times and subjected to Western blot analysis. Parts of the immunocomplexes were treated with 6 M Urea or boiled in 1% SDS for 4 min. Supernatants were diluted 20-fold with lysis buffer and reimmunoprecipitated using IgG or NEMO-conjugated beads and subjected to Western blot analysis using anti-Ubiquitin or anti-NEMO antibodies.

To examine whether stimulation with LPA or PMA plus Iono could induce the NEMO-associated polyubiquitination and whether this polyubiquitination is dependent on CARMA3, we stimulated Carma3^–/– MEFs (C3KO-Vector) or Carma3^–/– MEFs reconstituted with HA-tagged CARMA3 (C3KO-CARMA3) (Supplementary Fig. 2) with or without PMA plus Iono and then immunoprecipitated NEMO from these cells (Supplementary Fig. 3). However, we were unable to detect signal-induced polyubiquitination in the immunoprecipitated NEMO complex, either in the presence or absence of CARMA3 (Supplementary Fig. 3). These results suggest that the signal-induced, NEMO-associated polyubiquitination was either very transient or only a small amount of proteins were ubiquitinated.

TRAF6 is required for LPA-induced NF-B activation

It has been shown that TRAF6 and MALT1 may function as E3 ligases to induce polyubiquitination of NEMO (Sun et al. 2004; Zhou et al. 2004). In addition, TRAF6 deficiency also displays a similar defect of the neural tube closure as CARMA3 deficiency does (Lomaga et al. 2000), suggesting that TRAF6 and CARMA3 may function in the same signaling pathway leading to NF-B activation. To determine whether TRAF6 is involved in LPA-induced NF-B activation, we stimulated TRAF6-deficient MEF cells with LPA, PMA plus Iono, LPS, or TNF. Consistent with the role of TRAF6 in TLR but not TNF signaling pathway, LPS-induced NF-B was defective in TRAF6-deficient cells, whereas TNF effectively induced NF-B activation (Fig. 5A). Although LPA or PMA plus Iono effectively activated NF-B in wild-type cells, this activation of NF-B was completely defective in TRAF6-deficient cells (Fig. 5A), indicating that TRAF6 is required for GPCR-induced NF-B activation. Similar to the role of CARMA3 in IKK activation, LPA or PMA plus Iono could induce IKK phosphorylation in both wild-type and TRAF6-deficient cells (Fig. 5B). Together, these results indicate that similar to CARMA3, TRAF6 mediates LPA-induced NF-B activation through an IKK phosphorylation-independent mechanism.

View larger version (52K): [in this window] [in a new window]   Figure 5. TRAF6 and BCL10 play critical roles in LPA-induced NF-B activation. (A) Wild-type or TRAF6-deficient MEF cells were stimulated with or without LPA (10 µM) or PMA (10 ng/mL) plus Iono (100 ng/mL) for 60 min, or LPS (10 ng/mL) and TNF (1 ng/mL) for 30 min. Nuclear extracts were prepared from these cells and subjected to EMSA using 32P-labeled NF-B or Oct-1 probe. (B) Wild-type or TRAF6-deficient MEF cells were stimulated with or without LPA (10 µM) or PMA (10 ng/mL) plus Iono (100 ng/mL) for 10 or 20 min. Cell lysates were prepared from these cells and then subjected to Western blot analysis using the indicated antibodies. (C) Bcl10+/– or Bcl10–/– MEF cells were stimulated with or without LPA (10 µM) or PMA (10 ng/mL) plus Iono (100 ng/mL) for 10 or 20 min. Cell lysates were prepared from these cells and then subjected to SDS-PAGE and Western blot analysis using the indicated antibodies.

Bcl10 is not required for LPA-induced IKK/ phosphorylation

Earlier studies indicate that CARMA3 is physically associated with Bcl10 upon overexpression, and recent studies by us and others also indicate that Bcl10 is required for GPCR-induced NF-B activation (Klemm et al. 2007; McAllister-Lucas et al. 2007; Wang et al. 2007). To determine whether BCL10 is required for GPCR-induced IKK/ phosphorylation, Bcl10^+/– or Bcl10^–/– MEF cells were stimulated with or without LPA or PMA plus Iono. We found that although these stimulations failed to induce IB phosphorylation in Bcl10^–/– cells, they effectively induced IKK/ phosphorylation in both Bcl10^+/– and Bcl10^–/– cells (Fig. 5C). Thus, Bcl10 is also required for GPCR-induced NF-B activation but is not required for the signal-induced IKK/ phosphorylation.

CARMA3 functions as a common component in signaling pathways induced by other GPCRs, leading to activation of NF-B

To determine whether CARMA3 is also involved in signaling pathways induced by other GPCRs, wild-type or CARMA3-deficient MEF cells were stimulated with or without ET-1. ET-1 is another GPCR ligand that can induce NF-B activation (Purcell et al. 2001), and is a potent vasoconstrictor playing a critical role in cardiac hypertrophy (Kedzierski and Yanagisawa 2001). Although ET-1, as well as LPA or PMA plus Iono, effectively activated NF-B in Carma3^+/+ and Carma3^+/– MEF cells, it failed to activate NF-B in Carma3^–/– MEF cells (Fig. 6A). ET-1-induced NF-B activation was observed within 30 min and maintained up to 90 min (Fig. 6B), and pretreatment of the cells with cycloheximide did not inhibit ET-1-induced NF-B activation (Supplementary Fig. 4), suggesting that the NF-B activation induced by ET-1 was not due to the secondary stimulation from ET-1-induced cytokines. Similar to LPA stimulation, ET-1-induced phosphorylation of IB was completely defective in CARMA3-deficient cells (Fig. 6C) whereas ET-1-induced IKK phosphorylation was comparable in both wild-type and CARMA3-deficient MEF cells (Supplementary Fig. 5). Together, these results demonstrate that CARMA3 is required for NF-B activation induced by different GPCRs. Also similar to LPA-induced MAP kinase activation, ET-1-induced MAP kinase activation was comparable in wild-type and CARMA3-deficient cells (Fig. 6D). Therefore, these results demonstrate that GPCR-induced MAP kinase activation is not dependent on CARMA3.

View larger version (51K): [in this window] [in a new window]   Figure 6. CARMA3 mediates ET-1-induced NF-B activation. (A) Carma3+/+, Carma3+/–, and Carma3–/– MEF cells (1 x 106) were stimulated with or without ET-1 (10 nM) and LPA (10 µM) for 60 min, or PMA (10 ng/mL) plus Iono (100 ng/mL) or TNF (10 ng/mL) for 30 min. Nuclear extracts were prepared and then subjected to EMSA using the indicated probes. (B) Carma3+/+, Carma3+/–, and Carma3–/– MEF cells were stimulated with or without ET-1 (10 nM) for 15, 30, 60, and 90 min. Nuclear extracts were prepared and subjected to EMSA using the indicated probes. (C) Carma3+/+ and Carma3–/– MEF cells were stimulated with or without ET-1 (10 nM) or TNF (10 ng/mL) for indicated time points. Cell lysates were then subjected to SDS-PAGE and Western blot analysis using indicated antibodies. (D) Wild-type or CARMA3-deficient MEF cells were stimulated with ET-1 (10 nM) for various time points. Cell lysates were then subjected to SDS-PAGE and Western blot analysis using indicated antibodies.

It has been shown that many GPCRs, such as the receptors for LPA and ET-1, induce NF-B activation through G_q (Ye 2001). Thus, we expressed a constitutively active mutant of G_q, G_q(Q209L), which synergistically enhanced CARMA3-induced NF-B activation in HEK293 cells (Fig. 7A), supporting the hypothesis that G_q is functionally linked to CARMA3. To further demonstrate that G_q-induced NF-B activation is dependent on CARMA3, we infected wild-type or CARMA3-deficient MEF cells with lentiviral vector encoded G_q(Q209L). Although expression of G_q(Q209L) could induce NF-B activation in wild-type cells, it failed to activate NF-B in CARMA3-deficient cells (Fig. 7B, top panel). In contrast, expression of G_q(Q209L) induced comparable levels of AP-1 and MAP kinase activation (Fig. 7B [middle panel], C) in wild-type and CARMA3-deficient cells. Together, these results demonstrate that CARMA3 is selectively involved in G_q-mediated NF-B activation.

View larger version (22K): [in this window] [in a new window]   Figure 7. Gq-induced NF-B activation is dependent on CARMA3. (A) HEK293 cells were transfected with or without expression vectors encoding CARMA3 or Gq(Q209L) together with NF-B-dependent luciferase (firefly) reporter and EF1-dependent luciferase (Renilla) vectors. The transfected cells were harvested 20 h post-transfection and lysed in the lysis buffer. Luciferase activities were determined using the dual-luciferase reporter assay kit. NF-B-dependent activity was normalized by the constitutively active EF1-dependent luciferase activity. Data are the mean of triplicates from a representative experiment. (B) A constitutively active mutant of Gq, Gq(Q209L), was cloned into a lentiviral expression vector. The lentivirus encoding Gq(Q209L) was used to infect Carma3+/+ or Carma3–/– MEF cells. Nuclear and cytoplasmic extracts were prepared 72 h following the infection. Nuclear extracts were subjected to EMSA using indicated 32P-labeled probes. (C) Cytoplasmic extracts were subjected to SDS-PAGE and Western blot analysis using indicated antibodies.

	Discussion

Top Abstract Results Discussion Materials and methods Acknowledgments References

View larger version (20K): [in this window] [in a new window]   Figure 8. The working model for CARMA3-mediated NF-B activation in GPCR signal transduction pathway. GPCR-induced NF-B activation involves a signaling cascade of PKC–CARMA3–BCL10–MALT1–TRAF6 leading to polyubiquitination of the IKK complex, whereas a CARMA3-independent and PKC-dependent signal induces IKK phosphorylation by an unknown kinase in the GPCR signaling pathway. In the absence of CARMA3, GPCR-induced polyubiquitination of the IKK complex is defective, thereby resulting in a defective IKK complex.

Although it has been proposed that the IKK complex is ubiquitinated, there is not definite evidence showing NEMO is ubiquitinated. In this study we also observed that expression of CARMA3 induces polyubiquitination of the NEMO-containing complex, but we find that this ubiquitination is abolished following the treatment of the NEMO-containing complex with 6 M Urea or 1% SDS boiling. These results argue that the observed NEMO polyubiquitination is due to the polyubiquitination of an unknown protein(s) that is associated with NEMO. Therefore, it will be important to identify the ubiquitinated component(s) that associates with the IKK complex in the future studies.

It has been shown that TAK1, a MAP3K family member, is involved in NF-B activation induced by TNF and TLR (Sato et al. 2005; Shim et al. 2005), and TAK1 is proposed to phosphorylate IKK (C. Wang et al. 2001). Consistent with these studies, we found that TAK1 is required for antigen receptor-induced IKK/ phosphorylation (Shambharkar et al. 2007). However, LPA-induced IKK/ phosphorylation is not defective in TAK1-deficient MEF cells (data not shown). Therefore, it remains to be determined which kinase is responsible for GPCR-induced IKK/ phosphorylation.

In the TCR signaling pathway, PKC is activated following TCR stimulation, which induces phosphorylation of CARMA1 (Matsumoto et al. 2005; Sommer et al. 2005). The phosphorylated CARMA1 further recruits Bcl10 (Gaide et al. 2002; Hara et al. 2004; Wang et al. 2004), MALT1, and TRAF6 (Sun et al. 2004; Zhou et al. 2004). MALT1 and TRAF6 may then function as E3 ligases to further induce K63-linked polyubiquitination of NEMO, leading to activation of the IKK complex and subsequently NF-B (Sun et al. 2004; Zhou et al. 2004). CARMA3 contains the same structural domains as CARMA1, and associates with Bcl10 when overexpressed (McAllister-Lucas et al. 2001; L. Wang et al. 2001). We found that Bcl10, MALT1, and TRAF6 constitutively formed a complex in nonhematopoietic cells (data not shown). Therefore, we hypothesize that the Bcl10–MALT1–TRAF6 complex may function as a common signaling complex downstream from CARMA3 in GPCR signaling pathway (Fig. 8). Consistent with this hypothesis, we demonstrated that TRAF6 (Fig. 5) and Bcl10 (Wang et al. 2007) are required for GPCR-induced NF-B activation. Together, our results indicate that GPCR-induced NF-B activation involves a signaling cascade containing CARMA3, Bcl10, and TRAF6 in nonhematopoietic cells (Fig. 8). Since CARMA3 is only expressed in nonhematopoietic cells and its homologous protein, CARMA1, is expressed in hematopoietic cells, it will be interesting to investigate whether CARMA1 is required for GPCR-induced NF-B activation in hematopoietic cells.

It has been shown that the deficiency of >80 genes results in NTDs (Copp et al. 2003). Interestingly, mice deficient in IKK/IKK, Bcl10, or TRAF6 display a similar NTD phenotype as CARMA3-deficient mice (Copp et al. 2003), suggesting that the NTD observed in CARMA3-deficient mice is likely associated with impaired NF-B activation. Of note, mice deficient in CARMA3, Bcl10, and TRAF6 have a similar penetrance of the NTD phenotype, suggesting that these proteins may mediate the same signaling pathway by an unknown inducer, leading to activation of the IKK complex and NF-B in a subset of neural crest cells during neural tube development. Such induction of NF-B may be required for the survival of neural crest cells. Therefore, further studies will be needed to determine how CARMA3, Bcl10, and TRAF6 are involved in neural tube development and why only a certain percentage of mice deficient in these genes display the defect during their neural tube development.

Earlier studies suggest that both G_q and G_12/13 mediate GPCR-induced NF-B activation. G_q mediates NF-B activation induced by LPA and ET-1 (Ye 2001). Consistent with these observations, we can detect effective induction of NF-B upon overexpression of the constitutively active mutant of G_q, and we found that this induction of NF-B is dependent on CARMA3 (Fig. 7). In contrast, overexpression of the similar mutant of G_12/13 induced weak NF-B activation (data not shown). Therefore, it is difficult to draw a definite conclusion if G_12/13mediated NF-B activation is dependent on CARMA3. Nevertheless, our results indicate that NF-B activation induced by those GPCRs that utilize G_q is dependent on CARMA3.

In summary, our studies revealed a novel signaling cascade induced by GPCRs, in which GPCRs induce NF-B through CARMA3, which in turn regulates downstream signaling components such as Bcl10, TRAF6, and NEMO/IKK, leading to NF-B activation. Therefore, our results not only provide the genetic evidence that CARMA3 is required for GPCR-induced NF-B activation, but also provide a key link between the GPCR signaling pathway and the IKK complex. However, it will be important to determine how CARMA3 links to upstream signaling cascades in GPCR-induced signaling pathways and which kinase is responsible for IKK/ phosphorylation in future studies.

	Materials and methods

Top Abstract Results Discussion Materials and methods Acknowledgments References

 

Antibodies, plasmids, and reagents

Phospho-ERK1/2 (9101S), phospho-IB (9246L), phospho-JNK1/2 (9251L), JNK1/2 (9252), phospho-p65 (3036L), and G_q antibodies were from Cell Signaling. -Tubulin (D-10), ERK2 (C-14), IB (C21), Actin (C-2), IKK/ (H470), IKK (H-744), IKK (FL-419), Ubiquitin (P4D1), and Bcl10 (H-197) antibodies were from Santa Cruz Biotechnology, Inc. The peptide VRGRILQEQARLVWVEC, matching to the C terminus of human and mouse CARMA3, was used to immunize rabbits, and the corresponding antibodies were purified using affinity column conjugated with the same peptide. The expression plasmid encoding Gq(Q209L) was provided by Dr. Richard Ye (University of Illinois at Chicago, Chicago, IL). Electrophoretic mobility shift assay (EMSA) probes were from Promega. ET-1, PMA, and Iono were from Sigma. LPA was from Avanti. Tumor necrosis factor- was from Endogen.

Targeting vector and gene knockout

Carma3 genomic fragments were amplified from genomic DNA of mouse ES cells, and subcloned into the TK-containing pL2-Neo targeting vector (kindly provided by Dr. Hua Gu, Columbia University, New York) with the PGK-neomycin resistance cassette replacing Exon 3 of the Carma3 gene. The vector was electroporated into Sv129 ES cells and expanded in culture, and cells were selected under neomycin and gancyclovir. Genomic DNA was isolated from positive cells and screened using Southern blot. Two positive clones were used for injection into C57/Bl6 blastocysts, which were subsequently implanted into pseudo-pregnant C57/Bl6 females. Chimeric mice were intercrossed to generate heterozygous founder mice, which were further intercrossed to generate the Carma3^–/– animals. Carma3^–/– mice from both clones exhibited the same neural tube closure defect phenotype. Genomic DNA was isolated from mouse tails through proteinase K digestion and ethanol precipitation, followed by PCR using the following primers: 5'-CATTTTGC CTGGGAAACGC-3' (forward, Intron2), 5'-GGGTAGTAGAA TTCCAGGG-3' (reverse 1, Exon 3), and 5'-TGCCTGCTTGC CGAATATC-3' (reverse 2, Neo^r). Total RNA was isolated from MEFs using the RNeasy Protect Mini Kit (Qiagen), according to the manufacturer’s protocol. cDNA was prepared from the mRNA using the SuperScript III kit (Invitrogen), according to the manufacturer’s protocol. RT–PCR was conducted using the following primers: 5'-TGCTCAGCACCTACCGTTTC-3' (forward, Exon 2) and 5'-CCGAATTCTTCTCCTCGCTG-3' (reverse, Exon 6).

Mouse embryos and sections

To isolate embryos, pregnant females were sacrificed at different stages of pregnancy, and embryos were dissected out, separated from surrounding tissues, and photographed. For sections, embryos were fixed in 10% formalin, paraffin embedded, and sectioned at 4–6 µm diameters. Hematoxylin and eosin (H&E) stains were conducted according to standard protocols.

MEF preparation

MEF cells were prepared by removing day 12.5–13.5 embryos from mothers, separating them from the uterine wall and amniotic sac, and placing them in 0.25% trypsin. The entire embryo was then chopped using a razor blade, digested for 10 min at 37°C, and triturated through a Pasteur pipette. Cells were then split into two 10-cm dishes and grown to confluence in DMEM, and adherent cells were either frozen back as passage 0 or split again for use. TRAF6 KO MEF cells were kindly provided by Dr. Tak Mak (University of Toronto, Canada).

Generation of CARMA3-reconstituted MEF cells

Carma3^–/– MEF cells were transfected with a plasmid encoding E1A to immortalize as described previously (Flores et al. 2002). The immortalized Carma3^–/– MEF cells (C3KO) were further transfected with either HA-tagged mouse CARMA3 in the pCDNA3.1-Hygro vector or empty vector using the calcium phosphate precipitation technique. After 2 d, the cells were selected under hygromycin for 2 wk, and stable clones expressing CARMA3 were selected by Western blot analysis.

Western blot and immunoprecipitation

For detection of CARMA3, livers from adult mice were frozen in liquid nitrogen, followed by grinding with a mortar and pestle in RIPA buffer (150 mM NaCl, 10 mM Tris at pH 7.2, 0.1% SDS, 1% NP-40, 1% Deoxycholate, 5 mM EDTA, protease inhibitors). One-hundred micrograms of total protein were separated by SDS-PAGE and probed using anti-CARMA3 antibodies. For MAPK and IKK blots, 1 x 10⁶ MEFs were serum starved for 18 h, stimulated, and lysed in 100 µL of lysis buffer (150 mM NaCl, 50 mM HEPES at pH 7.4, 1 mM EDTA, 1% NP-40, protease inhibitors). Of the resulting lysates, 12–15 µL were subjected to SDS-PAGE and probed for the specific antibodies.

Electrophoretic mobility shift assay

MEF cells (1 x 10⁶) were starved for 18 h and stimulated for 30–60 min, and nuclear extracts were prepared. Nuclear extracts (5–10 µg) were then incubated with 1 x 10⁵ cpm of ³²P-labeled probes in 10 mM HEPES (pH 7.9), 40 mM NaCl, 1 mM EDTA, 4% glycerol, 3 µg Poly-dIC, and 0.5 mM DTT for 15 min at room temperature. The samples were then run on a nondenaturing polyacrylamide gel and exposed to film at –80°C.

MIP-2 ELISA

Wild-type or CARMA3-deficient MEF cells were first starved in the medium with 0.5% fetal calf serum for 18 h, and then stimulated with or without LPA (30 µM) or PMA (40 ng/mL) plus Iono (100 ng/mL) for another 20 h. The media from these cultures were collected and subjected to MIP-2 ELISA analysis according to the manufacturer’s instructions (Quantikine kit from R&D Systems, Inc).

Integrin-induced NF-B activation

Recombinant mouse OPN was from R&D Systems, Inc., H-Arg-Gly-Asp-Ser-OH (RGDS) was from Bachem, and poly-L-Lys (PLL) was from Sigma-Aldrich. PLL, OPN, or RGDS was resuspended in PBS and coated onto six-well dishes overnight at 4°C. The dishes were washed with PBS, and 2 x 10⁶ serum-starved MEF cells were plated onto the dishes for 2 h. For TNF stimulation, 10 ng/mL TNF were added to PLL-plated cells 30 min before harvest. Following stimulation, the cells were collected and lysed, and nuclear extracts were prepared. Nuclear extracts were then subjected to EMSA for the detection of NF-B activation.

	Acknowledgments

Top Abstract Results Discussion Materials and methods Acknowledgments References

 
We thank Drs. H. Gu, D. Wang, S. Ghosh, T. Mak, S. Lowe, and R. Ye for reagents. This work was partly supported by grants from the National Institutes of Health (GM065899 and AI050848) to X.L. X.L. is a Scholar of Leukemia and Lymphoma Society, and a recipient of the Investigator Award of Cancer Research Institute, Inc.

	Footnotes

 
⁵ These authors contributed equally to this work.

⁶ Corresponding author.

E-MAIL xllin{at}mdanderson.org; FAX (713) 794-0209.

Supplemental material is available at http://www.genesdev.org.

Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1502507

	References

Top Abstract Results Discussion Materials and methods Acknowledgments References

 
Bertin, J., Wang, L., Guo, Y., Jacobson, M.D., Poyet, J.L., Srinivasula, S.M., Merriam, S., DiStefano, P.S., and Alnemri, E.S. 2001. CARD11 and CARD14 are novel caspase recruitment domain (CARD)/membrane-associated guanylate kinase (MAGUK) family members that interact with BCL10 and activate NF-B. J. Biol. Chem. 276: 11877–11882.[Abstract/Free Full Text]

Biswas, D.K., Cruz, A.P., Gansberger, E., and Pardee, A.B. 2000. Epidermal growth factor-induced nuclear factor B activation: A major pathway of cell-cycle progression in estrogen-receptor negative breast cancer cells. Proc. Natl. Acad. Sci. 97: 8542–8547.[Abstract/Free Full Text]

Blonska, M., Pappu, B.P., Matsumoto, R., Li, H., Su, B., Wang, D., and Lin, X. 2007. The CARMA1–Bcl10 signaling complex selectively regulates JNK2 kinase in the T cell receptor-signaling pathway. Immunity 26: 55–66.[CrossRef][Medline]

Chen, Z.J. 2005. Ubiquitin signalling in the NF-B pathway. Nat. Cell Biol. 7: 758–765.[CrossRef][Medline]

Copp, A.J., Greene, N.D., and Murdoch, J.N. 2003. The genetic basis of mammalian neurulation. Nat. Rev. Genet. 4: 784–793.[CrossRef][Medline]

Cummings, R., Zhao, Y., Jacoby, D., Spannhake, E.W., Ohba, M., Garcia, J.G., Watkins, T., He, D., Saatian, B., and Natarajan, V. 2004. Protein kinase C mediates lysophosphatidic acid-induced NF-B activation and interleukin-8 secretion in human bronchial epithelial cells. J. Biol. Chem. 279: 41085–41094.[Abstract/Free Full Text]

Egawa, T., Albrecht, B., Favier, B., Sunshine, M., Mirchandani, K., O’Brien, W., Thome, M., and Littman, D. 2003. Requirement for CARMA1 in antigen receptor-induced NF-B activation and lymphocyte proliferation. Curr. Biol. 13: 1252–1258.[CrossRef][Medline]

Flores, E.R., Tsai, K.Y., Crowley, D., Sengupta, S., Yang, A., McKeon, F., and Jacks, T. 2002. p63 and p73 are required for p53-dependent apoptosis in response to DNA damage. Nature 416: 560–564.[CrossRef][Medline]

Gaide, O., Martinon, F., Micheau, O., Bonnet, D., Thome, M., and Tschopp, J. 2001. Carma1, a CARD-containing binding partner of Bcl10, induces Bcl10 phosphorylation and NF-B activation. FEBS Lett. 496: 121–127.[CrossRef][Medline]

Gaide, O., Favier, B., Legler, D.F., Bonnet, D., Brissoni, B., Valitutti, S., Bron, C., Tschopp, J., and Thome, M. 2002. CARMA1 is a critical lipid raft-associated regulator of TCR-induced NF-B activation. Nat. Immunol. 3: 836–843.[CrossRef][Medline]

Ghosh, S., May, M.J., and Kopp, E.B. 1998. NF-B and Rel proteins: Evolutionarily conserved mediators of immune responses. Annu. Rev. Immunol. 16: 225–260.[CrossRef][Medline]

Gilman, A.G. 1987. G proteins: Transducers of receptor-generated signals. Annu. Rev. Biochem. 56: 615–649.[CrossRef][Medline]

Hara, H., Wada, T., Bakal, C., Kozieradzki, I., Suzuki, S., Suzuki, N., Nghiem, M., Griffiths, E.K., Krawczyk, C., Bauer, B., et al. 2003. The MAGUK family protein CARD11 is essential for lymphocyte activation. Immunity 18: 763–775.[CrossRef][Medline]

Hara, H., Bakal, C., Wada, T., Bouchard, D., Rottapel, R., Saito, T., and Penninger, J.M. 2004. The molecular adapter Carma1 controls entry of IB kinase into the central immune synapse. J. Exp. Med. 200: 1167–1177.[Abstract/Free Full Text]

Hayden, M.S. and Ghosh, S. 2004. Signaling to NF-B. Genes & Dev. 18: 2195–2224.[Abstract/Free Full Text]

Juliano, R.L. 2002. Signal transduction by cell adhesion receptors and the cytoskeleton: Functions of integrins, cadherins, selectins, and immunoglobulin-superfamily members. Annu. Rev. Pharmacol. Toxicol. 42: 283–323.[CrossRef][Medline]

Jun, J., Wilson, L., Vinuesa, C., Lesage, S., Blery, M., Miosge, L., Cook, M., Kucharska, E., Hara, H., Penninger, J., et al. 2003. Identifying the MAGUK protein Carma-1 as a central regulator of humoral immune responses and atopy by genome-wide mouse mutagenesis. Immunity 18: 751–762.[CrossRef][Medline]

Karin, M. and Ben-Neriah, Y. 2000. Phosphorylation meets ubiquitination: The control of NF-B activity. Annu. Rev. Immunol. 18: 621–663.[CrossRef][Medline]

Kedzierski, R.M. and Yanagisawa, M. 2001. Endothelin system: The double-edged sword in health and disease. Annu. Rev. Pharmacol. Toxicol. 41: 851–876.[CrossRef][Medline]

Klemm, S., Zimmermann, S., Peschel, C., Mak, T.W., and Ruland, J. 2007. Bcl10 and Malt1 control lysophosphatidic acid-induced NF-B activation and cytokine production. Proc. Natl. Acad. Sci. 104: 134–138.[Abstract/Free Full Text]

Lomaga, M.A., Henderson, J.T., Elia, A.J., Robertson, J., Noyce, R.S., Yeh, W.C., and Mak, T.W. 2000. Tumor necrosis factor receptor-associated factor 6 (TRAF6) deficiency results in exencephaly and is required for apoptosis within the developing CNS. J. Neurosci. 20: 7384–7393.[Abstract/Free Full Text]