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During endochondral bone formation, chondrocyte hypertrophy represents a crucial turning point from chondrocyte differentiation to bone formation. Both parathyroid hormone-related protein (PTHrP) and histone deacetylase 4 (HDAC4) inhibit chondrocyte hypertrophy. Using multiple mouse genetics models, we demonstrate in vivo that HDAC4 is required for the effects of PTHrP on chondrocyte differentiation. We further show in vivo that PTHrP leads to reduced HDAC4 phosphorylation at the 14-3-3-binding sites and subsequent HDAC4 nuclear translocation. The Hdac4-KO mouse shares a similar but milder phenotype with the Pthrp-KO mouse, indicating the possible existence of other mediators of PTHrP action. We identify HDAC5 as an additional mediator of PTHrP signaling. While the Hdac5-KO mouse has no growth plate phenotype at birth, the KO of Hdac5 in addition to the KO of Hdac4 is required to block fully PTHrP action on chondrocyte differentiation at birth in vivo. Finally, we show that PTHrP suppresses myocyte enhancer factor 2 (Mef2) action that allows runt-related transcription factor 2 (Runx2) mRNA expression needed for chondrocyte hypertrophy. Our results demonstrate that PTHrP inhibits chondrocyte hypertrophy and subsequent bone formation in vivo by allowing HDAC4 and HDAC5 to block the Mef2/Runx2 signaling cascade. These results explain the phenotypes of several genetic abnormalities in humans.

Although thyroid hormone is a known stimulator of erythropoietic differentiation, severe anemia is sometimes observed in patients with hyperthyroidism and this mechanism is not fully understood. The aim of this study was to investigate the effect of triiodothyronine (T3) on hemin-induced erythropoiesis. Human erythroleukemia K562 cells were used as an erythroid differentiation model. Cell differentiation was induced by hemin and the effect of pre-incubation with T3 (0.1 to 100 nM) was analyzed by measuring the benzidine-positive rate, hemoglobin content, CD71 expression (transferrin receptor), and mRNA expression for transcription factors related to erythropoiesis and thyroid hormone receptors (TRs). Hemin, a promoter of erythroid differentiation, increased the levels of mRNAs for TRα, TRβ, and retinoid X receptor α (RXRα), as well as those for nuclear factor-erythroid 2 (NFE2), GATA-binding protein 1 (GATA1) and GATA-binding protein 2 (GATA2). Lower concentrations of T3 had a stimulatory effect on hemin-induced hemoglobin production (1 and 10 nM), CD71 expression (0.1 nM), and α-globin mRNA expression (1 nM), while a higher concentration of T3 (100 nM) abrogated the stimulatory effect on these parameters. T3 at 100 nM did not affect cell viability and proliferation, suggesting that the abrogation of erythropoiesis enhancement was not due to toxicity. T3 at 100 nM also significantly inhibited expression of GATA2 and RXRα mRNA, compared to 1 nM T3. We conclude that a high concentration of T3 attenuates the classical stimulatory effect on erythropoiesis exerted by a low concentration of T3 in hemin-induced K562 cells.

Thyroid hormone stimulates erythropoietic differentiation. However, severe anemia is sometimes seen in patients with hyperthyroidism, and the mechanisms have not been fully elucidated. Bone marrow is comprised about 2–8 % oxygen, and the characteristics of hematopoietic stem cells have been shown to be influenced under hypoxia. Hypoxia-inducible factor-1 is a critical mediator of cellular responses to hypoxia and an important mediator in signal transduction of thyroid hormone [triiodothyronine (T3)]. The aim of this study was to investigate the effect of T3 on erythropoiesis under hypoxia mimicking physiological conditions in the bone marrow. We maintained human erythroleukemia K562 cells under hypoxic atmosphere (2 % O2) and examined their cellular characteristics. Compared to that under normal atmospheric conditions, cells under hypoxia showed a reduction in the proliferation rate and increase in the hemoglobin content or benzidine-positive rate, indicating promotion of erythroid differentiation. T3 had no effect on hypoxia-induced erythroid differentiation, but significantly inhibited activin A/erythroid differentiation factor-induced erythroid differentiation. Moreover, GATA2 mRNA expression was suppressed in association with erythroid differentiation, while T3 significantly diminished that suppression. These results suggest that T3 has a direct suppressive effect on erythroid differentiation under hypoxic conditions.