Calcineurin and NFAT4 Induce Chondrogenesis*

Nuclear factor of activated T-cells (NFAT) and calcineurin are essential regulators of immune cell and mesenchymal cell differentiation. Here we show that elevated intracellular calcium induces chondrogenesis through a calcineurin/NFAT signaling axis that activates bone morphogenetic protein (BMP) expression. The calcium ionophore, ionomycin, induced chondrogenesis through activation of calcineurin. The calcineurin substrate, NFAT4, also induced chondrogenesis and chondrocyte gene expression. Significantly, the BMP antagonist, noggin, or dominant negative BMP receptors blocked the effects of elevated intracellular calcium on chondrogenesis. This suggested that calcineurin/NFAT4 activates BMP expression. Consistent with this, BMP2 gene expression was increased by ionomycin and suppressed by the calcineurin inhibitor, cyclosporine A. Furthermore, activated NFAT4 induced BMP2 gene expression. These results have important implications for the effects of NFATs during development and adaptive responses.

The vertebrate skeleton develops from a template that is initially constructed of cartilage. The formation of this template is controlled by a series of interdependent steps that supply spatial information and morphogenic cues that direct mesenchymal cell condensation and differentiation in a process known as chondrogenesis. Mesenchymal cells derived from the lateral plate mesoderm condense to form the rudiments of the appendicular skeleton. The processes that control the condensation of mesenchyme are poorly understood. Cell-cell adhesion certainly contributes to mesenchymal cell condensation as demonstrated in studies showing that homotypic interactions of N-cadherin are required during chondrogenesis (1,2). In addition, a number of extracellular signaling molecules have been proposed to regulate chondrogenesis by regulating both condensation and differentiation. WNTs (3), fibroblast growth factors (4), sonic hedgehog (5), and bone morphogenetic proteins (BMPs) 1 (6 -8) all contribute to chondrogenesis. Increasing evidence indicates that BMPs have a central role in this process. For example, loss of function experiments using dominant negative BMP receptors suppresses chondrogenesis during chicken limb development (9,10). Conversely, overexpression of BMPs or activated BMP receptors dramatically augments chondrogenesis during limb development (10,11). These data suggest that elucidating pathways that control BMP expression will be vital to understanding chondrogenesis.
Intracellular calcium ([Ca 2ϩ ] i ) is a universal intracellular signal that intersects with many signaling molecules to regulate gene expression (12). However, the role of intracellular calcium in chondrogenesis is virtually uncharacterized. Elevations of [Ca 2ϩ ] i activate a number of signaling cascades, including the calcineurin/nuclear factor of activated T-cell (NFAT) pathway (13). Prolonged elevations of [Ca 2ϩ ] i activate the phosphatase calcineurin, through interaction with Ca 2ϩ :calmodulin (14). In turn, calcineurin dephosphorylates a set of substrates, including the transcription factors, NFATs (15). Dephosphorylation of NFATs results in translocation of the protein from the cytoplasm to the nucleus (16). In the nucleus, NFATs activate gene expression in cooperation with other transcriptional regulators (17,18). NFATs are a family of four transcription factors, NFAT1(p, c2), NFAT2(c, c1), NFAT3, and NFAT4(x,c3). Although well known for their ability to regulate cytokine gene expression in immune cells (15), NFATs are broadly expressed and have essential functions outside of the immune system (18 -21). The transcriptional targets of NFATs outside of immune cells, however, are incompletely characterized. Here we report that elevation of [Ca 2ϩ ] i induce chondrogenesis using a pathway requiring calcineurin, NFAT4, and BMP expression. In this pathway BMP2 expression is induced by activated calcineurin/NFAT. Subsequently, BMP2 induces chondrogenesis through an autocrine loop.

EXPERIMENTAL PROCEDURES
Cells and Cell Culture-RCJ3.1C5.18 cells (22) were generously provided by Jane Aubin. Cells were maintained at 37°C, 5% CO 2 in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (Hyclone), penicillin (100 units/ml), and streptomycin (100 g/ml). Limb bud mesenchymal cells were grown in 1:1 Dulbecco's modified Eagle's medium and Ham's F-12 containing the same supplements. Limb bud mesenchyme was derived from e11.5 day mouse embryos as described previously (23). Briefly, single cell suspensions were obtained from isolated limb buds following digestion with 0.1% trypsin:EDTA (Invitrogen) and 2 mg/ml collagenase IA (Sigma) for 30 min at 37°C with agitation. Proteolysis was inhibited with an equal volume of bovine serum, and the suspension was passed through a 70-m cell strainer. Cells were washed twice with growth medium, suspended at 1-5 ϫ 10 6 cells/ml, and plated in 20-l aliquots. Four hours after plating the wells were flooded with growth medium. Medium was changed every 3 days.
Transient Transfections-Cells were transfected in triplicate using a modified calcium phosphate precipitate. Briefly, cells were plated 24 h prior to transfection at 8 ϫ 10 4 cells/ml in a 12-well plate (800 l/well). The medium was replaced 4 h before transfection. 7.5 g of each pNFATluc (Stratagene) construct and 0.7 g of pCS␤-gal were diluted to a final volume of 20 l with water. To that was added 355 l of 0.26 M CaCl 2 and 375 l of 2ϫ BBS (50 mM BES, 280 mM NaCl, 1.5 mM Na 2 HPO 4 ). After precipitation at room temperature for 10 min, 80 l of the precipitate was added directly to the cells. Cells were incubated at 37°C for 20 h, at which point the precipitate was washed off and replaced with complete medium for 24 h. Thereafter cells were treated with ionomycin (1.0 M), cyclosporine A (2.5 g/ml), or both for 24 h. Cell extracts were prepared, and luciferase and ␤-galactosidase activity was analyzed by chemiluminesence.
Alcian Blue Staining-Cultures were washed with phosphate-buffered saline, fixed for 30 min with 10% buffered formalin, equilibrated with a 3% solution of acetic acid, and then stained for 20 min with 5% Alcian blue (Sigma), pH 3.0. Unbound dye was removed by extensive washing with distilled water. Quantification of cartilage differentiation was determined by counting the number of Alcian blue staining nodules.
Northern Blotting-RNA was prepared from cells using an RNeasy (Qiagen) kit according to the manufacturer's instructions. 10 -20 g of total RNA was loaded to a low formaldehyde-agarose gel. Samples were fractionated and blotted to nitrocellulose membranes (Gelman). Membranes were hybridized with [ 32 P]dCTP-lableled aggrecan (p1355 plasmid provided by Y. Yamada) or GAPDH (pTRI-GAPDH, Ambion) cDNA fragments. DNA probes were labeled with a Prime-It kit (Stratagene) according to the manufacturer's specifications. Relative levels of gene expression were determined by phosphorimage analysis using a Molecular Dyanamics Storm. For each sample, the level of mRNA was normalized to a GAPDH or 18 S rRNA internal control.
Western Blotting-Cells were lysed in 1% SDS, 10 mM dithiothreitol, 50 mM Tris, pH 6.8; DNA sheared by passage through a 25-gauge needle; and denatured 10 min at 95°C. Proteins were resolved on a 5% SDS-PAGE gel, transferred to polyvinylidene difluoride membrane, and detected by enhanced chemifluorescence as suggested by the manufacturer (Pierce). Anti-NFAT4 antibody (Santa Cruz, clone F-1) was used at a 1:100 dilution in blocking solution (5% milk proteins in phosphatebuffered saline) and incubated overnight at 4°C. The secondary antibody (goat anti-mouse conjugated to horseradish peroxidase, Cappel) was diluted 1:2000 in blocking solution and incubated 1 h at 25°C.
Adenoviruses-Adenoviral constructs were made as described previously (21). Recombinant adenoviruses Smad6, dominant negative ALK6KR, and dominant negative ALK3KR (24) were generously supplied by M. Fujii. Adenoviruses were titered by limiting dilution. Infection at m.o.i. of 100 ensured that greater that 95% of the cells were infected as assessed by LacZ staining or indirect immunofluorescence of the transduced protein.
Indirect Immunofluorescence-Limb bud cultures plated on coverslips were washed with phosphate-buffered saline and then fixed with 4% paraformaldehyde in phoshate-buffered saline. Samples were microwaved in 10 mM citrate buffer, pH 6.0, until the buffer was 95°C. This was repeated three times. The monoclonal antibody II-II6B3 developed by Thomas Linsenmayer was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences. The anti-type X collagen antibody pXNC1 was generously provided by Greg Lunstrum (Shriners Hospital, Portland, OR). Primary antibodies were diluted 1:500 in blocking buffer (5% goat serum, 1% bovine serum albumin, 0.1% Triton X-100 in phosphate-buffered saline) and incubated overnight at 4°C. Goat anti-mouse rhodamine (Jackson ImmunoResearch) and donkey ant-rabbit fluorescein (Jackson Immu-noResearch) were diluted 1:250 in blocking solution and incubated with the sample for 2 h at room temperature.
Statistical Analysis-Statistical comparisons were done using an unpaired Student's t test. The null hypothesis was rejected for p Ͻ 0.05.

RESULTS
Chondrogenesis can be modeled by assessing the development of cartilage from limb bud mesenchyme derived from mouse embryos. In this assay limb buds are excised from 11.5 days postcoitus embryos, cell suspensions prepared by proteolysis, and the suspension was plated at high density in cell culture. To determine the effects of intracellular calcium on the development of cartilage, we investigated the effects of the calcium ionophore, ionomycin, on the differentiation of limb bud mesenchyme. Interestingly, we found that ionomycin significantly activated chondrogenesis (Fig. 1A). Ionomycin produced a 2.5-fold increase (p ϭ 0.002) in the number of Alcian blue staining nodules of cartilage. Increased chondrogenesis was also evidenced by indirect immunofluoresecnce for collag-ens type II (Fig. 1B) and type X (Fig. 1C). Significantly, cyclosporine A (CsA) inhibited cartilage development both in the presence and absence of ionomycin (Fig. 1A). Because CsA inhibits calcineurin, this finding implicated the calcium-calmodulin dependent phosphatase as an essential regulator of chondrogenesis, even in the absence of the calcium ionophore. To further examine the effects of intracellular calcium and calcineurin on chondrogenesis, we studied the effects of ionomycin on gene expression in the chondrocytic mesenchymal cell line RCJ3.1C5.18 (RCJ). These cells differentiate into chondrocytes (22) and can simulate chondrogenesis ex vivo. We studied the effect of ionomycin on aggrecan expression, because aggrecan is an early-expressed and essential cartilage-specifc proteoglycan. Consistent with the effects of ionomycin on limb bud mesenchyme, we found that ionomycin strongly induced aggrecan gene expression ( Fig. 2A). 2 M ionomycin caused a 15-fold increase in gene expression. Additionally, CsA completely inhibited the induction of aggrecan expression. Furthermore, adenoviral transduction of the polypeptide inhibitor of calcineurin, CAIN (25), suppressed the induction of aggrecan by ionomycin (Fig. 2B). These data indicate that calcineurin activates chondrocyte differentiation. To directly test this, we studied the effect of an activated calcineurin on aggrecan gene expression in RCJ cells. A truncated, calcium-calmodulin-independent calcineurin A (26) induced aggrecan gene expression when transduced into RCJ cells using a recombinant adenovirus (Fig. 2C). Similarly, adenoviral transduction of activated calcineurin into limb bud mesenchyme augmented chondrogenesis (Fig. 2D). Activated CnA adenovirus produced a 1.7-fold increase (p ϭ 0.01) in Alcian blue-stained cartilage nodules, relative to ␤-galactosidase control.
These results suggested that certain calcineurin substrates induce chondrogenesis. Because the calcineurin substrate, nu-  (27), we hypothesized that other NFAT family members induce chondrogenesis in response to calcium. We examined NFAT4 because it is broadly expressed and has been found to regulate mesenchymal cells (21,28,29). Western blots showed NFAT4 protein in both RCJ cells and limb bud mesenchyme (Fig. 3A). In limb bud mesenchyme, we observed hypophoshorylated forms of NFAT4 in response to ionomycin, consistent with NFAT4 activation. In addition, NFAT activity was found in RCJ cells following iononmycin treatment, as indicated by increased transcriptional activity of an NFAT-luciferase reporter plasmid (Fig. 3B). We then asked whether a constitutively active, calcineurin-independent NFAT4 (⌬NFAT4) could induce aggrecan gene expression. This protein lacks the amino-terminal regulatory region and has constitutive nuclear localization and transactivating properties (30,31). Interestingly, adenoviral transduction of ⌬NFAT4 increased aggrecan gene expression in RCJ cells 2.5fold (Fig. 3C). Induction of aggrecan expression was not observed with a control ␤-galactosidase adenovirus. Also, transduction of the ⌬NFAT4 into limb bud mesenchyme augmented chondrogenesis as evidenced by a 2-fold increase (p ϭ 0.005) in the number of Alcian blue staining cartilage nodules (Fig. 3D).
The molecular pathways used by calcineurin to activate chondrogenesis are unknown. However, certain growth factors/ cytokines are critical regulators of mesenchymal cell differentiation. BMP family members are essential for limb bud chondrogenesis (10). We therefore asked whether the effects of ionomycin on aggrecan gene expression require BMP signaling. First the effects of the BMP antagonist, noggin, were exam-ined. Interestingly, noggin effectively blocked (70% inhibition) the induction of aggrecan by ionomycin (Fig. 4A). In addition, adenoviral transduction of dominant negative, kinase-inactive BMP receptors ALK6KR or ALK3KR (24) caused a 80 and 60% inhibition of aggrecan gene expression in response to ionomycin (Fig. 4B), respectively. Furthermore, Smad6, which inhibits BMP signaling, repressed ionomycin-induced aggrecan expression by 50% (Fig. 4B). These results clearly demonstrate that BMP signaling is required for aggrecan induction is response to ionomycin.
We showed that 1) the effect of ionomycin on chondrocyte differentiation requires both activation of calcineurin and BMP signaling, and 2) the calcineurin substrate NFAT4 induces chondrogenesis and chondrocyte gene expression. These data suggest the hypothesis that calcineurin-dependent pathways induce BMP expression through NFAT4. To test this, we asked whether ionomycin induces BMP expression through a calcineurin dependent pathway. We discovered that ionomycin caused a 2-2.7-fold increase in BMP2 expression in RCJ cells (Fig. 4C). Moreover, BMP2 induction was dependent on calcineurin activity as evidenced by repression by cyclosporine A (Fig. 4C). ⌬NFAT4 also induced BMP2 expression (2-fold increase, Fig. 4D), supporting the hypothesis that calcineurin regulates BMP2 expression through NFAT activation. Cumulatively, these data are consistent with the model of Fig. 5. Elevation of [Ca 2ϩ ] i activates calmodulin, which in turn activates calcineurin. Calcineurin dephosphorylates NFAT4 leading to nuclear translocation. In the nucleus, NFATs activate the expression of the BMP2 gene. BMP2 then induces aggrecan gene expression by an autocrine or paracrine pathway.

DISCUSSION
BMPs are potent regulators of mesenchymal cell differentiation. Therefore, understanding the pathways that induce BMP expression is vital to understanding processes like chondrogenesis or chondrocyte differentiation. However, in vertebrates relatively little is known of the pathways and transcription factors that directly induce BMP gene expression. Perhaps the best understood pathway that directly induces BMP gene expression is the hedgehog pathway. This pathway has been studied principally in Drosophila where the transcription factor cubitus interruptus induces dpp (the Drosophila homolog of BMP2/4) following the binding of hedgehog to its receptor, patched (32,33). Mammalian homologs of cubitus interruptus (Gli1-3) have been identified (34), and when expressed in Drosophila they can similarly induce dpp expression (35). DNA promoter sequences for mouse and human BMP2 and BMP4 have been identified (36 -38). However, specific transactivators of the promoter, other than Gli (39), have not been reported. Therefore, except for the established paradigm of hedgehog/ patched/cubitus interruptus, little is known of the regulation of BMP expression by specific transcription factors. We describe a novel pathway that induces BMP2 gene expression following calcineurin and NFAT activation. Interestingly, calcineurin-dependent NFATs (NFAT1, -2, -3, and 4) are not found in Drosophila or Caenorhabditis elegans, indicating that this pathway is restricted to higher eukaryotes.
[Ca 2ϩ ] i can be increased by the action of growth factors/ cytokines, ion channels, and basic cell biological processes such as assembly and dissociation of cell-cell or cell-cell matrix contacts. This places the calcineurin/NFAT pathway at a focal point of signal transduction pathways that may be required to coordinate chondrogenesis. Interestingly, NFATs may also be required for the maintenance of articular cartilage and suppression of juxta-articular osteophytes. Mice homozygous for a mutation in the NFAT1 gene develop an age-dependent arthropathy (27). The articular cartilage of these animals shows degenerative features suggestive of accelerated osteoarthritis. In addition, there is pathological cartilagenous differentiation in the soft tissues adjacent to the joint. These findings suggest that there may be distinct effects of NFAT1 on articular chondrocytes and extra-articular soft tissues. In articular chondrocytes, NFAT1 may be required for chondrocyte homeostasis, whereas in the adjacent soft tissues NFAT1 may be required to prevent aberrant differentiation.
The pathway of induction of BMP expression by NFATs may be germane to other incompletely understood functions of NFATs. For example, null mutations of NFAT2 in mice are lethal due to the absence of semilunar cardiac valves (20,40). By contrast, Mash6 (Smad6) null mice have hypertrophic semilunar valves (41). Because Smad6 inhibits BMP signaling (42,43), the hypertrophic valves of Mash6Ϫ/Ϫ mice should be the consequence of excessive BMP signaling. If NFAT2 activates BMP expression during valvulogenesis, then nfat2Ϫ/Ϫ mice should have a phenotype opposite to that of Mash6 null mice. In fact, this is the observed phenotype. In support of a role of NFAT2 in regulating BMP expression, we have also observed that activated NFAT2 can induce cartilage gene expression (data not shown).  ] i . Calmodulin binds to and activates calcineurin, which dephosphorylates NFATs. Following dephosphorylation, NFATs move to the nucleus where BMP2 gene expression is induced. BMP2 is synthesized, binds to BMP receptors, and thereby activates signaling pathways that activate aggrecan gene expression.