4C). This nuclear protein/DNA complex was more abundant when cells treated with TSH (Fig. 4C). To investigate whether PKA is also involved in increased CERB-DNA binding activity stimulated by TSH, PKA inhibitor H89 was added, and the faint gel bands were found. In addition, ChIP assay showed that TSH markedly increased pCREB binding capacity in comparison to the control (P < 0.001), whereas H89 dramatically down-regulated this activation by TSH (P = 0.019 versus control). Likewise, knockdown of TSHR by RNAi inhibited TSH-induced CREB activation (P = 0.002 versus TSH) (Fig. 4D). Taken together,

these results suggest that TSH-induced elevation of cellular cAMP levels activates PKA. PKA in turn phosphorylates and activates CREB, which transcriptionally AP24534 activates RO4929097 HMGCR. To further investigate the role of TSH in the regulation of HMGCR, we pursued in vivo studies in rats. Circulating T4 was reduced to an undetectable level whereas serum TSH was dramatically elevated, and this was accompanied by a significant increase in plasma TC (P = 0.041) in Tx rats compared with the Sh rats (Fig. 5A, Table 1). After treatment with T4, endogenous TSH levels in Tx rats were reduced to low levels. Moreover, administration of T4 to Tx rats reduced the elevated serum TC to levels similar to those observed in Sh rats. In addition,

hepatic tissue proteins from Tx and Sh rats were analyzed for HMGCR and LDLR proteins, respectively. A significant increase (P = 0.004) in the HMGCR and a significant decrease (P = 0.038) in the LDLR in Tx rats relative to Sh animals were observed (Fig. 5B). We then administered exogenous TSH to these Tx rats at 0.05, 0.3, or 1.5 IU/rat daily for 7 days while they received daily T4, respectively. There was no significant difference in the serum T4 levels in the group of Tx rats receiving only exogenous www.selleck.co.jp/products/BIBF1120.html T4 compared with the Tx rats receiving both exogenous T4 and TSH (P > 0.05), whereas the serum TSH levels statistically increased in the group of Tx rats receiving exogenous TSH compared

with the Tx rats receiving only exogenous T4 (Fig. 6A, upper). Furthermore, we observed a dose-dependent increase in serum TC after administration of exogenous TSH, although this increase marginally failed to reach statistical significance (P > 0.05) when comparing the group of Tx rats receiving only exogenous T4 with the group of Tx rats receiving both exogenous T4 and TSH (Fig. 6A, upper). However, in the same group of Tx rats constantly receiving exogenous T4, a significant increase in serum TC was observed after TSH injection compared with before injection (Fig. 6A, lower). No significant difference in serum calcium, phosphorus, or liver function (alanine aminotransferase and aspartate aminotransferase) was observed among the different groups of animals.