Inhibition of RAR activity enhances chondrogenesis through a Sox9-dependent mechanism
Previously, the continued expression of RARα activity in transgenic mice was found to inhibit the chondroprogenitor-to-chondroblast transition. Likewise, inhibition of RARα using the subtype-specific antagonist, AGN194301, induced differentiation in primary limb mesenchymal cultures earlier than normal, resulting in a substantial increase in the number of cartilage nodules that form in these cultures. This induction of cartilage formation was confirmed by the AGN194301-induced increase in expression of cartilage-specific genes such as Col2a1
(Weston et al., 2000
). Given that Sox9 has been shown previously to be important in regulating the expression of Col2a1
, we analyzed the effects of RARα antagonism on Sox9
expression and activity in an attempt to further understand the mechanism whereby retinoid signaling regulates chondroblast differentiation.
To follow endogenous Sox9 activity in primary mesenchymal cells, a reporter-based approach was used in which cells were transiently transfected with pGL3(4X48), a reporter containing four repeats of a Sox9 binding site from the first intron of Col2a1.
The RARα-specific antagonist, AGN194301 (301), induced a concentration-dependent increase in reporter activity, whereas at-RA and the RARα-specific agonist, AGN193836 (836), attenuated reporter activity ( A). Interestingly, when cells were treated with the RAR pan-antagonist, AGN194310 (310), concentrations as low as 10 nM induced Sox9 reporter activity greater than the maximal response elicited by higher doses of 301. The maximal response to the pan antagonist was ~530% induction at 50 nM, whereas the greatest induction of Sox9 reporter activity by the RARα-specific antagonist was ~280% at 1 μM, a concentration at which this antagonist affects ligand binding to other RAR subtypes (Weston et al., 2000
). Similar to RAR antagonism, the reduction in reporter activity caused by a pan-agonist such as at-RA was more pronounced than that induced by the RARα-specific agonist, 836. at-RA reduces reporter activity to 53% at 5 nM, whereas in response to a much higher dose of 836 (1 μM) reporter activity is reduced only to 64% of control. Together, these results indicate that a loss in activity of at least two or more RARs is more efficient at inducing cartilage differentiation than inhibition of the RARα subtype alone.
Figure 1. Inhibition of RAR activity enhances Sox9 activity and expression. Activation of the retinoid receptors in primary limb mesenchymal cultures with either at-RA or the RARα-specific agonist AGN193836 (836) attenuates activity of the pGL3(4X48) Sox9 (more ...)
Interestingly, the effects of RAR modulation on Sox9 activity are opposite to that of the effects of each compound on activity of a retinoic acid responsive reporter (pW1-βRARE3-Luc) in primary limb mesenchymal cells ( B). For instance, at-RA activates the RARE reporter to a greater extent than 836, whereas reporter activity is attenuated by 310 more effectively than by 301. Thus, activation of the Sox9-responsive region of Col2a1 appears to be very closely associated with the status of RAR activity. This close association is reflected in the response of primary cultures to treatment with each compound ( C). Treatment with either at-RA, the RARα agonist, or with the antagonists for 4 d affects the formation of cartilage nodules in a manner that would be predicted from their effects on Sox9 reporter activity. More specifically, at-RA is a more potent inhibitor of cartilage nodule formation than 836, whereas the increase in nodule formation can be observed at a lower concentration of the pan-antagonist, (10 nM 310) compared with the RARα-specific antagonist (1 μM 301). Together, these results further validate the utility of the Sox9 reporter assay to indirectly measure the status of chondroblast differentiation, and more importantly they highlight the significant role of RAR-mediated signaling in regulating expression of the chondroblast phenotype.
The enhanced Sox9 reporter activity caused by RAR inhibition is due, in part, to an increase in the expression of Sox9 mRNA, since treatment of primary cultures with 1 μM 301 results in an precocious increase in Sox9 expression ( D). There is a noticeable increase in Sox9 mRNA from 2-d cultures treated with 301, but this increase over control cultures is much less pronounced by days 4 and 6. Thus, inhibition of RAR activity appears to induce an early transient upregulation of Sox9 mRNA that presumably contributes to the enhanced Sox9 reporter activity seen in response to the same compound.
To confirm the influence of RAR activity on chondroblast differentiation, we introduced modified versions of the RARs or RXRs into primary limb mesenchymal cultures to follow their effect on Sox9 reporter activity. To examine the effect of RAR activation without agonist addition, constitutively active versions of RARα and RXRα were used by fusing the acidic activation domain of VP16 to the COOH terminus of RAR and RXR referred to as RARαVP16 and RXRαVP16, respectively (Underhill et al., 1994
). Here we confirmed the ability of these modified receptors to potently activate an RARE reporter in the absence of an exogenous agonist, since cotransfection of RARαVP16 and RXRαVP16 induced RARE reporter activity by 15- and 17-fold, respectively, in the absence of agonist ( A).
Figure 2. Sox9 transactivation of Col2a1 is inversely associated with RAR activity. To further study the influence of retinoid receptor activity on chondrogenesis, constructs containing constitutively active receptors (RARαVP16 and RXRαVP16) or (more ...)
In addition to modifying RARE activity with constitutively active versions of the retinoid receptors, dominant negative versions of the receptors (dnRARα and dnRXRα) were also generated and transfected into primary limb mesenchymal cultures. These dominant negative derivatives are COOH-terminal truncations of RARα and RXRα that retain their ability to bind DNA and ligand but lack the AF-2 transactivation function (Damm et al., 1993
; Feng et al., 1997
). When cotransfected into primary cultures, both dnRARα and dnRXRα are effective at completely blocking activity of the RARE reporter ( B). Cotransfection of the modified receptors affects Sox9 reporter activity in a manner that is inversely proportional to their ability to trans-activate βRARE3
-tk-Luc. Both the RARαVP16 and RXRαVP16 inhibit Sox9 reporter activity, whereas the dnRARα and dnRXRα potently activate this reporter (). Interestingly, the activation induced by cotransfection with dnRARα is more dramatic than that elicited by any other factor studied to date, including those examined by our laboratory and those reported previously. Similar to the results using receptor agonists and antagonists, these studies demonstrate a strong influence of retinoid receptor activity on chondrogenesis.
Chondrogenic response to RAR inhibition requires 48 bp enhancer elements within Col2a1 and is specific to chondrogenic cells
To closely examine the contribution of RAR inhibition to Sox9 activity, reporters with varying sensitivities to Sox9 were used to follow their response to the dnRARα. Of four reporters analyzed, 4X48-p89 and pGL3(4X48), which demonstrate the greatest sensitivity to Sox9 ( A), also exhibit the greatest response to dnRARα ( B). In contrast, pGL3(−89+6), a reporter containing only the minimal Col2a1 promoter with no 48 bp Sox9 binding sites, exhibits no activity in response to Sox9 and is unaffected by the dnRARα (). A reporter containing two tandem repeats of a larger intron-1 segment of Col2a1 (including Sox9 binding sites) along with a promoter fragment is only mildly sensitive to Sox9 and is activated to a much lesser extent by dnRARα compared with the 4X48-containing reporters. These results demonstrate a direct relationship between inhibition of RAR signaling and Sox9 activity.
Figure 3. Sox9 binding sites are essential for dnRARα-induced reporter activity. To examine the contribution of Sox9 to the effects of RAR inhibition, reporters with varying sensitivities to Sox9 were used to follow their response to the dnRARα. (more ...)
Despite the induction of Sox9 reporter activity elicited by dnRARα in other cells with chondrogenic capacity, such as dedifferentiated rat articular chondrocytes and C5.18 chondroprogenitor cells, activity of Sox9 reporter activity is not noticeably affected in COS P7 cells (). Given that COS P7 cells are nonchondrogenic, these results suggest that the Sox9 reporter induction caused by RAR inhibition may be restricted to cells with chondrogenic capacity.
Figure 4. Induction of Sox9 activity by dnRARα is observed in other chondrogenic cells. The induction of Sox9 reporter activity by dnRARα in different cells compared with vector-transfected (−) controls is shown. The effect of dnRARα (more ...)
Chondrogenesis requires histone deacetylase-mediated gene repression
Transcriptional regulation by the retinoid receptors depends for the most part on ligand availability. In the absence of ligand, RAR/RXR heterodimers bind to and repress the transcription of various target genes. Receptor-mediated repression is due to association with nuclear complexes containing corepressors (nuclear receptor corepressor [N-CoR] and SMRT) and histone deacetylases (HDACs) (Nagy et al., 1997
). Trichostatin A (TSA) is a Streptomyces
metabolite that specifically inhibits histone deacetylases leading to hyperacetylation of histones and other proteins (Finnin et al., 1999
). To date, TSA has been shown to act as a potent inducer of differentiation in many cell types, some of which are also induced to differentiate by treatment with RA. Interestingly, chondroprogenitors, which in contrast to most cell types do not differentiate in response to RA, also respond uniquely to TSA as indicated by both a dose-dependent decrease in Sox9 reporter activity ( A) and in cartilage nodule formation ( C) in response to a TSA-induced increase in RARE reporter activity ( B). TSA also attenuates the 310-induced increase in Sox9 reporter activity and nodule formation (). The inhibitory effects of TSA on chondrogenesis are achieved at relatively low concentrations of TSA, since higher concentrations have been used to induce differentiation of many cell types including NIH3T3 cells and acute promyelocytic leukemia blasts (Sugita et al., 1992
; Ferrara et al., 2001
). Moreover, the well-characterized ability of TSA to inhibit IL-2 gene expression was found to have an IC50
of 73 nM (Koyama et al., 2000
), which is greater than the highest concentration (10 nM) used here. Thus, these results demonstrate an important requirement for HDAC-mediated gene repression in chondroblast differentiation.
Figure 5. Histone deacetylase-mediated gene repression is required for chondrogenesis. The effects of TSA on Sox9 reporter activity in the presence or absence of AGN194310 (A) and on pW1-βRARE3tkLuc (B) were analyzed. TSA attenuated Sox9 reporter activity (more ...)
To further examine the importance of nuclear corepressors in chondroblast differentiation, we examined the ability of a dominant negative version of N-CoR, pCMX-G/N-CoR(2174–2453), to modulate Sox9 reporter activity. This construct lacks the HDAC interaction domain and contains the nuclear hormone receptor interaction domain of N-CoR, a region similar to that of SMRT, which was shown recently to disrupt nuclear corepressor function (Koide et al., 2001
). Consistent with these activities, the pCMX-G/N-CoR(2174–2453) inhibited the ability of the antagonists and the dnRAR to decrease RARE reporter activity (unpublished data). Expression of pCMX-G/N-CoR(2174–2453) alone led to an ~50% decrease in basal Sox9 reporter activity. Moreover, coexpression of pCMX-G/N-CoR(2174–2453) completely inhibited the stimulatory effects of 301 and 310 and repressed the effect of the dnRAR on the Sox9 reporter ( D). These results suggest that active repression by RARs is required for chondroblast differentiation and that this repression requires deacetylase activity.
RAR inhibition activates the p38 MAPK and PKA pathways
To elucidate the mechanism whereby a loss in RAR activity leads to enhanced Sox9 activity, pathway profiling vectors were used to uncover signal transduction pathways that act downstream of retinoid signaling. Various reporters containing reiterated enhancer sequences were transiently cotransfected into primary cultures with a dnRARα. Cotransfection with the dnRARα was used as it is a potent constitutive repressor that consistently induces high Sox9 activity in primary cells. The luciferase-based reporters used contained response elements for activating protein-1 (pAP-1-TA-Luc), cAMP (pCRE-TA-Luc), nuclear factor of κB cells (pNFκB-TA-Luc), nuclear factor of activated T cells (pNFAT-TA-luc), serum, (pSRE-TA-Luc), glucocorticoids (pGRE-TA-Luc), and interferons (pISRE-TA-Luc). Each vector contained the reiterated response elements upstream of a TATA box and the luciferase gene. Interestingly, when cotransfected with a dnRARα the only reporters appreciably affected (greater than twofold increases) were pCRE-TA-Luc and pAP-1-TA-Luc. Cotransfection with dnRARα enhanced activity of these reporters by greater than fourfold (), indicating that RAR inhibition may result in activation of pathways upstream of CRE and AP-1 responses.
Figure 6. The p38 MAPK pathway and the PKA pathway are activated in response to RAR inhibition. Reporters containing a cAMP response element (pCRE-TA-Luc) or activator protein-1 response element (pAP-1-TA-Luc) are both activated in response to cotransfection with (more ...)
The PKA pathway is a predominant pathway through which genes containing a cAMP-response element (CRE) are activated. When activated through various stimuli, PKA phosphorylates CRE binding protein (CREB), which binds to and activates genes containing cAMP response elements. Accordingly, cotransfection of pCMV-PKA dramatically enhances activation of pCRE-TA-Luc (unpublished data). Given that a pCRE-TA-Luc is activated in cells transfected with a dnRARα, we tested the ability of this modified receptor to induce activation of CREB. A chimeric trans-activator protein containing CREB fused to the DNA binding domain of the yeast transcriptional activator GAL4 (pFA-CREB) was transiently transfected into cells with a luciferase reporter containing a reiterated GAL4 DNA binding element. Thus, by monitoring the activity of the pG5-Luc reporter the activation of FA-CREB was indirectly followed. Cotransfection of pCMV-PKA into the primary cultures induced an ~40-fold increase in pG5-Luc ( C). Cotransfection with dnRARα enhanced FA-CREB-induced transactivation of pG5-Luc by approximately sixfold.
In addition to the PKA pathway, we investigated potential mechanisms that may underlie the activation of AP-1 by dnRARα. Activating protein-1 collectively refers to dimeric transcription factors composed of Jun, Fos, or activating transcription factor (ATF) subunits. Surprisingly, a dominant negative version of Fos (A-Fos), which substantially diminishes pAP-1-TA-Luc reporter activity, was found to have no noticeable effect on activity of the Sox9 reporter (unpublished), suggesting that the induction of pAP-1-TA-Luc by dnRARα does not involve activation of Jun/Fos dimers. Moreover, constitutively active versions of kinases within the MAPK pathways were tested for their ability to modulate Sox9 transactivation. Of the kinases known to be upstream of AP-1 activation, only a constitutively active version of MKK6 (MKK6E) consistently led to increased Sox9 reporter activity. The predominant targets of MKK6 appear to be the p38 mitogen-activated protein kinase (MAPK) isoforms. When phosphorylated, p38 phosphorylates and activates several targets including the AP-1 component ATF2. As a positive control, MKK6E was cotransfected into cells and found to induce activity of pAP-1-TA-Luc (unpublished data). Given that ATF2 has been shown to bind to AP-1 response elements, we used the pG5-Luc reporter to measure the activity of FA-ATF2, a chimeric of ATF2 and the DNA binding domain of GAL4. Cotransfection of dnRARα induced an increase in FA-ATF2 activation of pG5-Luc that was almost as robust as the induction by MKK6E ( D).
Further support for the role of p38 MAPK and PKA in chondroblast differentiation comes from the reduction in Sox9 reporter activity caused by the p38 MAPK inhibitor SB202190 and the PKA inhibitor H89 (). These inhibitors also attenuated the induction of Sox9 reporter activity by dnRARα and by 301 (). Consistent with this, the inhibitors at 10 μM inhibited the formation of cartilage nodules in untreated () and 301-treated cultures (unpublished data) compared with untreated cultures ().
Figure 7. Inhibition of p38 and PKA prevents chondrogenesis. In the presence of 5 or 10 μM SB202190, there is a decrease in Sox9 reporter activity compared with untreated controls (A). SB202190 also attenuates the chondrogenic response to AGN194301 and (more ...)
Activation of ATF2 and CREB induces Sox9 transactivation response
The studies described above suggest that the suppression of RAR activity leads to activation of the p38 MAPK and PKA signaling pathways. Phosphorylation of ATF2 and CREB is reflective of activation of p38 MAPK and PKA signaling pathways, respectively. To further investigate a possible role for these signaling pathways in the activation of Sox9, factors involved in these pathways were transiently transfected into the mesenchymal cells along with the Sox9 reporter. Transient transfection of a constitutively active version of MKK6 (MKK6E) induces an approximate threefold activation of FA-ATF2 ( A). When transfected along with p38α or p38β, MKK6E is able to induce FA-ATF2 activity by ~13- and 14-fold, respectively, and even more so with the two isoforms together. However, p38α and p38β alone or in combination have no noticeable effect on Sox9 activity. The ability of each expression plasmid to induce activation of FA-ATF2 is directly proportional to their influence on Sox9 reporter activity ( B), with a >4.5-fold activation by cotransfection with MKK6E along with p38α and p38β. Similarly, Sox9 is activated by the catalytic subunit of PKA, which potently enhances FA-CREB activity. However, the induction of Sox9 activity by PKA is relatively mild given the level of FA-CREB activation elicited by PKA. These results demonstrate the relevance of activation of the p38 and PKA pathways by dnRARα, since each pathway has the potential to induce Sox9 transactivation of Col2a1.
Figure 8. Activation of ATF2 or CREB induces Sox9 transactivation response. The effects of different components of the p38 signaling cascade on ATF2 induction were analyzed. Transient transfection with MKK6E induces ATF2 activity greater than twofold; however, (more ...) Sox9
DNA binding and hence its transcriptional activity has been shown to be induced by PKA-mediated phosphorylation of serines 64 and 181 (Huang et al., 2000
). Specifically, PKA phosphorylation of serine 181 in Sox9 was found to occur in chondrocytes of the prehypertrophic zone in response to parathyroid hormone-related peptide (Huang et al., 2000
). To determine if the same phosphorylation event occurs here in response to RAR antagonism, we compared the ability of dnRARα to induce Sox9 reporter activity in the presence of a cotransfected vector containing wtSox9 versus a mutant Sox9 in which serine 181 was replaced with alanine (Sox9-181A). In the absence of exogenous Sox9, Sox9 reporter activity is increased ~4.5 fold by activation of the PKA pathway using pCPT-cAMP (500 μM) in comparison to an ~9-fold increase by coexpression of dnRARα. Cotransfection with wt Sox9 or Sox9-181A increased reporter activity ~100 fold, and this was further increased, albeit slightly in each case (<1.5 fold), by the addition of pCPT-cAMP or by coexpression of PKAc or a dnRARα and decreased by the addition of H89 ( A; unpublished data). There is no significant difference in the activity of Sox9 versus Sox9-181A, suggesting that phosphorylation of serine 181 is not required for Sox9 activity during chondroblast differentiation. As mentioned, immunolocalization studies detected Ser181-phosphorylated Sox9 in prehypertrophic chondrocytes. However, the cells used here are chondroprogenitors, and thus, Sox9 activity may be regulated through distinct posttranslational modifications within each cell type. To ensure that the mutant Sox9 functions in a manner consistent with that reported previously (Huang et al., 2000
), wtSox9 and Sox9-181A were transfected into COS P7 cells in the presence or absence of an expression vector for the catalytic subunit of PKA. COS P7 cells were originally used to identify Ser181 as the PKA phosphorylation site, and as expected the ability of PKAc to activate Sox9 in these cells is almost completely blocked by the Ser181 mutation ( B). Thus, similar to earlier reports Ser181 of Sox9 appears to be required for increased activation of Sox9 by PKA in these cells, but this is clearly not the case in the limb mesenchymal cells used in this study.
Figure 9. Regulation of Sox9 expression and transcriptional activity by PKA. Sox9 expression and activity were measured in response to manipulation of the PKA signaling pathway. In the absence of exogenous Sox9, addition of pCPT-cAMP (500 μM) or cotransfection (more ...)
To determine if modulation of PKA activity affects the expression of Sox9
transcripts, real-time quantitative PCR was used to measure their relative expression levels in comparison to rRNA
expression were increased by more than twofold in response to a 2-d treatment with pCPT-cAMP (500 μM) and decreased by more than twofold in response to H89 (10 μM) ( C). A similar increase in Col2a1
expression by activation of PKA in limb mesenchymal cultures has been reported previously (Kosher et al., 1986
). Therefore, our results suggest that PKA regulates Sox9 activity during chondroblast differentiation by influencing Sox9
expression levels and not through phosphorylation of Ser181. However, a posttranslational role for PKA modulation of Sox9 cannot be entirely excluded, since cotransfected Sox9s do exhibit slightly increased activity in the presence of cAMP or cotransfected PKA.
The influence of p38 MAPK and PKA signaling pathways on chondrogenesis is best demonstrated by their ability to rescue the decrease in Sox9 activity induced by RARαVP16 (). Although Sox9 activity is only partially restored by cotransfection with RARαVP16 and MKK6E, transfection of each isoform in combination with MKK6E results in levels of Sox9 activity that are almost as high as those obtained in the absence of RARαVP16. Not surprisingly, MKK6E cotransfected with both isoforms of p38 (which causes the most pronounced activation of ATF2) results in a complete rescue of Sox9 activity ( A). Similarly, PKAc can almost completely rescue the effects of RARαVP16 ( B). These results are not due to modulation of RARαVP16, since neither MKK6E or PKAc inhibited RARαVP16 induction of an RARE reporter (unpublished data).
Figure 10. Activation of ATF2 or CREB can rescue the effects of RARαVP16. The ability of ATF2 and CREB to reverse the effects of RARαVP16 on Sox9 reporter activity was analyzed. MKK6E can partially prevent the inhibitory response of RARαVP16 (more ...)