Phosphorylation of SOX9 by Cyclic AMP-Dependent Protein Kinase A Enhances SOX9's Ability To Transactivate aCol2a1 Chondrocyte-Specific Enhancer

Abstract

Sox9 is a high-mobility-group domain-containing transcription factor required for chondrocyte differentiation and cartilage formation. We used a yeast two-hybrid method based on Son of Sevenless (SOS) recruitment to screen a chondrocyte cDNA library and found that the catalytic subunit of cyclic AMP (cAMP)-dependent protein kinase A (PKA-C␣) interacted specifically with SOX9. Next we found that two consensus PKA phosphorylation sites within SOX9 could be phosphorylated by PKA in vitro and that SOX9 could be phosphorylated by PKA-C␣ in vivo. In COS-7 cells cotransfected with PKA-C␣ and SOX9 expression plasmids, PKA enhanced the phosphorylation of wild-type SOX9 but did not affect phosphorylation of a SOX9 protein in which the two PKA phosphorylation sites (S 64 and S 211 ) were mutated. Using a phosphospecific antibody that specifically recognized SOX9 phosphorylated at serine 211, one of the two PKA phosphorylation sites, we demonstrated that addition of cAMP to chondrocytes strongly increased the phosphorylation of endogenous Sox9. In addition, immunohistochemistry of mouse embryo hind legs showed that Sox9 phosphorylated at serine 211 was principally localized in the prehypertrophic zone of the growth plate, corresponding to the major site of expression of the parathyroid hormone-related peptide (PTHrP) receptor. Since cAMP has previously been shown to effectively increase the mRNA levels of Col2a1 and other specific markers of chondrocyte differentiation in culture, we then asked whether PKA phosphorylation could modulate the activity of SOX9. Addition of 8-bromo-cAMP to chondrocytes in culture increased the activity of a transiently transfected SOX9-dependent 48-bp Col2a1 chondrocyte-specific enhancer; similarly, cotransfection of PKA-C␣ increased the activity of this enhancer. Mutations of the two PKA phosphorylation consensus sites of SOX9 markedly decreased the PKA-C␣ activation of this enhancer by SOX9. PKA phosphorylation and the mutations in the consensus PKA phosphorylation sites of SOX9 did not alter its nuclear localization. In vitro phosphorylation of SOX9 by PKA resulted in more efficient DNA binding. We conclude that SOX9 is a target of cAMP signaling and that phosphorylation of SOX9 by PKA enhances its transcriptional and DNA-binding activity. Because PTHrP signaling is mediated by cAMP, our results support the hypothesis that Sox9 is a target of PTHrP signaling in the growth plate and that the increased activity of Sox9 might mediate the effect of PTHrP in maintaining the cells as nonhypertrophic chondrocytes.The transcription factor SOX9 contains a high-mobilitygroup (HMG)-type DNA-binding domain that shows 50% identity to that of the mammalian testis-determining factor SRY (35) and a transcription activation domain located at the carboxyl terminus of the molecule (27, 31). During embryonic development, expression of Sox9 parallels that of the gene for type II collagen (Col2a1) in all chondrocyte progenitors and chondrocytes (27,36,37). In addition, Sox9 is also expressed in gonadal ridges in ...

Functional Gene Screening System Identified TRPV4 as a Regulator of Chondrogenic Differentiation

“…Some genes listed in Table 1 are known or thought to be involved in chondrogenesis. For example, protein kinase A, which had the strongest effect on SOX9-dependent transcription in our assay (Table 1), has been found to induce phosphorylation of SOX9 on serine residues Ser 64 and Ser 181 and to enhance the activity of SOX9 with respect to the Col2a1 chondrocyte-specific enhancer (26). Our screening also identified SOX5 and SOX6, both of which are well known to be required in chondrogenesis.…”

Oxygen Tension Regulates Chondrocyte Differentiation and Function during Endochondral Ossification

“…However, it was not up-regulated but rather was down-regulated by hypoxia. Sox9 transcriptional activity has been reported to be enhanced by cAMP-dependent protein kinase A serine/threonine phosphorylation (63). We found that phosphorylation of Sox9 was not promoted but was suppressed by hypoxia (Fig.…”

Acetylation reduces SOX 9 nuclear entry and ACAN gene transactivation in human chondrocytes

“…Like many other regulators, we speculated SOX9 is prone to posttranslational modifications which may impact its activity during adulthood and thereby possibly leading to its limited transcriptional capacity. For example, SOX9 was shown to be phosphorylated by PKA on serine 211 rendering its augmented DNA binding to the 18‐bp COL2A1 enhancer sequence (Huang et al ., 2000). TIP60 was first discovered to target SOX9 for acetylation by Hattori et al .…”