Younho Han

Acetylation of Sirt2 by p300 attenuates its deacetylase activity.

Histone deacetylases (HDACs) are subdivided into three classes--HDAC I, HDAC II, and Sir2. Sirt proteins are mammalian members of the Sir2 family of NAD+ (nicotinamide adenine dinucleotide)-dependent protein deacetylases. The balance between acetylation and deacetylation of histone and non-histone proteins, regulated by protein acetyltransferases and deacetylases, affects the expression of genes involved in a variety of cellular processes. In addition, HDAC1 is acetylated and regulated by p300, a transcriptional co-activator with protein acetyltransferase activity, suggesting that protein acetyltransferases and deacetylases they control the activities of each other. Although the regulation of HDAC1 by p300 is well characterized, the relationship between Sir2 homologs and p300 is not understood. Here, we report that p300 interacts with Sirt2, a member of the Sir2 family, and triggers the acetylation and subsequent down-regulation of the deacetylation activity of Sirt2, and that the acetylation of Sirt2 by p300 relieves the inhibitory effect of Sirt2 on the transcriptional activity of p53. These observations demonstrate that p300 can inactivate Sirt2 by acetylation and that p300 may regulate the activity of p53 indirectly through Sirt2 in addition to its direct modification of p53.

Acetylation of histone deacetylase 6 by p300 attenuates its deacetylase activity.

Protein acetyltransferases and deacetylases affect the activities of each other. This is well documented by the acetylation and inhibition of HDAC1 by p300, a transcriptional co-activator with protein acetyltransferase activity. However, the relationship between HDAC6 and p300 is poorly understood. HDAC6 is a class II histone deacetylase and differs from other members of HDAC family in that it contains two HDAC domains and an ubiquitin-binding motif. HDAC6 is a microtubule-associated deacetylase. It predominantly deacetylates non-histone proteins, including alpha-tubulin, and regulates cell motility. Here, we report that p300 interacts with and acetylates HDAC6 resulting down-regulation of HDAC6 deacetylase activity. Furthermore, we provide evidences that acetylation of HDAC6 by p300 inhibits tubulin deacetylation and suppression of Sp1 transcriptional activity by HDAC6. Our results demonstrate that p300 can inactivate HDAC6 by acetylation, and that p300 may regulate the activity of Sp1 indirectly through HDAC6 in addition to its direct modification of Sp1.

Melatonin promotes osteoblast differentiation by regulating Osterix protein stability and expression

Although the biological role of melatonin in osteogenic differentiation has been suggested, the mechanism of osteoblast differentiation remains unclear. Thus, the present study investigated the underlying molecular mechanisms based on osteoblast-specific transcription factors. We found that melatonin enhanced BMP-4-induced osteogenic differentiation and increased the expression of osteogenic markers, especially Osterix, which is an essential transcription factor for the differentiation of preosteoblasts into mature osteoblasts in the late stage of osteoblast differentiation. Melatonin treatment increased the expression of Osterix during osteoblast differentiation and stabilized its expression by the inhibition of ubiquitin-proteasome-mediated degradation of Osterix, leading to up-regulated Osterix transcriptional activity on the osteogenic promoter and promoting alkaline phosphatase activity and bone mineralization. Furthermore, treatment with protein kinase A (PKA) inhibitor H89 and protein kinase C (PKC) inhibitor Go6976 blocked the melatonin-induced transcriptional activity and phosphorylation of Osterix, indicating that melatonin regulates Osterix expression via the PKA and PKC signaling pathways. Overall, these findings suggest that melatonin directly regulates the late stage of osteoblast differentiation by enhancing Osterix expression; this provides further evidence of melatonin as a potent agent for treating osteoporosis.

Yin Yang 1 is a multi-functional regulator of adipocyte differentiation in 3T3-L1 cells.

Yin Yang 1 (YY1) is an ubiquitously distributed transcription factor that belongs to the GLI-Kruppel class of zinc finger proteins. The mechanism by which YY1 regulates adipocyte differentiation remains unclear. In this study, we investigated the functional role of YY1 during adipocyte differentiation. During the early stage, YY1 gene and protein expression was transiently downregulated upon the induction of differentiation, however, it was consistently induced during the later stage. YY1 overexpression decreased adipocyte differentiation and blocked cell differentiation at the preadipocyte stage, while YY1 knockdown by RNA interference increased adipocyte differentiation. YY1 physically interacted with PPARγ (Peroxisome proliferator-activated receptor gamma) and C/EBPβ (CCAAT/enhancer-binding protein beta) respectively in 3T3-L1 cells. Through its interaction with PPARγ, YY1 directly decreased PPARγ transcriptional activity. YY1 ectopic expression prevented C/EBPβ from binding to the PPARγ promoter, resulting in the downregulation of PPARγ transcriptional activity. These results indicate that YY1 repressed adipocyte differentiation by repressing the activity of adipogenic transcriptional factors in 3T3-L1 cells.

Protein kinase A phosphorylates and regulates the osteogenic activity of Dlx5.

Dlx5 transcription factor plays important roles in osteoblast differentiation and its transcription is regulated by many osteogenic signals including BMP-2. Recent studies suggest that the function of Dlx5 is also regulated post-translationally by protein kinases such as p38 and CaMKII. Protein kinase A (PKA) is involved in several steps of osteoblast differentiation and its activity has been shown necessary, yet not sufficient, for BMP-induced osteoblast differentiation. PKA is a ubiquitous cellular kinase that phosphorylates serine and threonine residues(s) of target proteins. In this study, we investigated the potential regulation of Dlx5 function by PKA in osteoblast differentiation. We found that PKA phosphorylates Dlx5 and that PKA activation increases the protein stability, osteogenic activity and transcriptional activity of Dlx5. We also found that BMP-2 increases the protein level of Dlx5 in a PKA activity-dependent manner. These results suggest that PKA activity enhances the osteogenic function of Dlx5, at least in part, through protein stabilization and that BMP-2 regulates the osteogenic function of Dlx5, at least in part, through PKA.

PKA-Mediated stabilization of FoxH1 negatively regulates ERα activity

Estrogen receptor α (ERα) mediates the mitogenic effects of estrogen. ERα signaling regulates the normal growth and differentiation of mammary tissue, but uncontrolled ERα activation increases the risk to breast cancer. Estrogen binding induces ligand-dependent ERα activation, thereby facilitating ERα dimerization, promoter binding and coactivator recruitment. ERα can also be activated in a ligand-independent manner by many signaling pathways, including protein kinase A (PKA) signaling. However, in several ERα-positive breast cancer cells, PKA inhibits estrogen-dependent cell growth. FoxH1 represses the transcriptional activities of estrogen receptors and androgen receptors (AR). Interestingly, FoxH1 has been found to inhibit the PKA-induced and ligand-induced activation of AR. In the present study, we examined the effects of PKA activation on the ability of FoxH1 to represses ERα transcriptional activity. We found that PKA increases the protein stability of FoxH1, and that FoxH1 inhibits PKA-induced and estradiol-induced activation of an estrogen response element (ERE). Furthermore, in MCF7 cells, FoxH1 knockdown increased the PKA-induced and estradiol-induced activation of the ERE. These results suggest that PKA can negatively regulate ERα, at least in part, through FoxH1.

Prolyl isomerase Pin1 regulates the osteogenic activity of Osterix.

Osterix is an essential transcription factor for osteoblast differentiation and bone formation. The mechanism of regulation of Osterix by post-translational modification remains unknown. Peptidyl-prolyl isomerase 1 (Pin1) catalyzes the isomerization of pSer/Thr-Pro bonds and induces a conformational change in its substrates, subsequently regulating diverse cellular processes. In this study, we demonstrated that Pin1 interacts with Osterix and influences its protein stability and transcriptional activity. This regulation is likely due to the suppression of poly-ubiquitination-mediated proteasomal degradation of Osterix. Collectively, our data demonstrate that Pin1 is a novel regulator of Osterix and may play an essential role in the regulation of osteogenic differentiation.

Cbl-b and c-Cbl negatively regulate osteoblast differentiation by enhancing ubiquitination and degradation of Osterix☆

E3 ubiquitin ligase Cbl-b and c-Cbl play important roles in bone formation and maintenance. Cbl-b and c-Cbl regulate the activity of various receptor tyrosine kinases and intracellular protein tyrosine kinases mainly by regulating the degradation of target proteins. However, the precise mechanisms of how Cbl-b and c-Cbl regulate osteoblast differentiation are not well known. In this study, we investigated potential targets of Cbl-b and c-Cbl. We found that Cbl-b and c-Cbl inhibit BMP2-induced osteoblast differentiation in mesenchymal cells. Among various osteogenic transcription factors, we identified that Cbl-b and c-Cbl suppress the protein stability and transcriptional activity of Osterix. Our results suggest that Cbl-b and c-Cbl inhibit the function of Osterix by enhancing the ubiquitin-proteasome-mediated degradation of Osterix. Taken together, we propose novel regulatory roles of Cbl-b and c-Cbl during osteoblast differentiation in which Cbl-b and c-Cbl regulate the degradation of Osterix through the ubiquitin-proteasome pathway.

Estrogen Receptor α Regulates Dlx3-Mediated Osteoblast Differentiation.

Estrogen receptor α (ER-α), which is involved in bone metabolism and breast cancer, has been shown to have transcriptional targets. Dlx3 is essential for the skeletal development and plays an important role in osteoblast differentiation. Various osteogenic stimulators and transcription factors can induce the protein expression of Dlx3. However, the regulatory function of ER-α in the Dlx3 mediated osteogenic process remains unknown. Therefore, we investigated the regulation of Dlx3 and found that ER-α is a positive regulator of Dlx3 transcription in BMP2-induced osteoblast differentiation. We also found that ER-α interacts with Dlx3 and increases its transcriptional activity and DNA binding affinity. Furthermore, we demonstrated that the regulation of Dlx3 activity by ER-α is independent of the ligand (estradiol) binding domain. These results indicate that Dlx3 is a novel target of ER-α, and that ER-α regulates the osteoblast differentiation through modulation of Dlx3 expression and/or interaction with Dlx3.

Src enhances osteogenic differentiation through phosphorylation of Osterix

Osterix, a zinc-finger transcription factor, is required for osteoblast differentiation and new bone formation during embryonic development. The c-Src of tyrosine kinase is involved in a variety of cellular signaling pathways, leading to the induction of DNA synthesis, cell proliferation, and cytoskeletal reorganization. Src activity is tightly regulated and its dysregulation leads to constitutive activation and cellular transformation. The function of Osterix can be also modulated by post-translational modification. But the precise molecular signaling mechanisms between Osterix and c-Src are not known. In this study we investigated the potential regulation of Osterix function by c-Src in osteoblast differentiation. We found that c-Src activation increases protein stability, osteogenic activity and transcriptional activity of Osterix. The siRNA-mediated knockdown of c-Src decreased the protein levels and transcriptional activity of Osterix. Conversely, Src specific inhibitor, SU6656, decreased the protein levels and transcriptional activity of Osterix. The c-Src interacts with and phosphorylates Osterix. These results suggest that c-Src signaling modulates osteoblast differentiation at least in part through Osterix.