In this report, we show for the first time that a rexinoid antagonist increases TSHβ mRNA in thyrotropes using an in vitro and in vivo model. The rexinoid antagonist affects TSHβ mRNA levels, at least in part, directly at the level of gene transcription. These data strongly suggest that the ‘TSH setpoint’ in thyrotropes is likely influenced by an endogenous rexinoid.
Vitamin A affects thyroid function and the main effect on thyrotrope function appears to be through an RXR-mediated or rexinoid pathway (Golden 2007
; Haugen 2004
; Morley 1980
; Sharma 2006
; Sherman 1999
). We have previously shown that mice lacking the RXRγ isotype have higher serum TSH and T4 levels than wild-type littermates, indicating that this isotype is important in rexinoid signaling in the thyrotrope (Brown et al 2000
). We also demonstrated high levels of the RXRγ1 isotype in mouse pituitaries and no compensatory increase in the RXRα and RXRβ isotypes in our RXRγ knock-out animals. These data suggest that rexinoid signaling and RXRγ play a role in the hypothalamic-pituitary-axis and that an endogenous rexinoid may be an important regulator of TSH in thyrotropes.
Macchia and colleagues demonstrated that mice lacking thyroid hormone receptor TRβ, which is critical for thyroid hormone signaling in the thyrotrope, still exhibited suppression of TSH with the rexinoid AGN194204 (Macchia 2002
). Our group and others further showed that the effects of thyroid hormone and rexinoids on TSHβ promoter activity were mediated by different regions of the TSHβ promoter (Breen 1997
; Sharma 2006
). Together, these data indicate that thyroid hormone and rexinoids have distinct and independent effects on TSH regulation in the thyrotrope.
It is now clear that exogenous rexinoids suppress TSH in human, animal and in vitro
retinoic acid (9-cis RA) was the first identified endogenous rexinoid, but it is believed that 9-cis
RA likely does not play a significant role in physiologic rexinoid signaling (Wolf 2006
). Others have shown that unsaturated fatty acids including oleic acid, linoleic acid, arachadonic acid and docosahexaenoic acid are putative endogenous rexinoids in multiple tissues (de Urquiza 2000
; Goldstein 2003
We chose a reverse pharmacology approach to explore rexinoid signaling in thyrotropes using both cell culture (TαT1 cells) and intact organism mouse models. We hypothesized that if an endogenous rexinoid contributed to TSH regulation in the thyrotrope, treatment with a rexinoid antagonist would raise TSHβ mRNA and TSH levels in these models. The rexinoid antagonist LG101208 significantly increased TSHβ mRNA levels in TαT1 cells and antagonized the effect of the exogenous rexinoid, LG100268. One potential mechanism could be an indirect effect through a decrease in deiodinase type 2 (mD2), which would lead to decreased local T3 production and increased TSHβ mRNA. The rexinoid antagonist did not decrease mD2 and actually increased mRNA levels at 48 hours, suggesting that the effect of LG101208 was not through effects on mD2 levels. We further showed that the effect of rexinoid antagonist on raising TSHβ mRNA levels in our thyrotrope model was lost in the presence of charcoal-stripped serum, indicating that a lipophilic endogenous rexinoid is present in serum. Charcoal treatment of serum efficiently removes many free fatty acids (Chen 1967
), which may serve as endogenous rexinoids. This is further supported by experiments showing that hypothyroid serum, which does not remove lipophilic compounds like free fatty acids, does not abrogate this rexinoid effect. These experiments do not rule out the possibility that the endogenous rexinoid is an intracellular fatty acid whose level is controlled by signaling from another serum factor.
It appears that rexinoid agonist suppression of TSHβ mRNA and rexinoid antagonist elevation of TSHβ mRNA in TαT1 thyrotropes occurs primarily at the level of gene transcription since both of these effects were abrogated in the presence of DRB, an RNA polymerase II inhibitor. Our group and others have previously shown in humans and animal models that rexinoids suppress TSH and T4 levels and that this effect appears to occur at the level of the pituitary and not the hypothalamus (Golden 2007
; Liu 2002
; Sharma 2006
; Sherman 1999
). We therefore predicted that inhibition of rexinoid signaling by the antagonist LG101208 would increase serum TSH and T4 levels and increase pituitary TSHβ mRNA levels in mice with an intact hypothalamic-pituitary-thyroid (HPT) axis. After three days of rexinoid antagonist treatment, mice had significantly higher serum T4 levels and slightly higher TSH levels (although not statistically significant) suggesting an altered ‘TSH setpoint’ in these animals with an intact HPT axis. We further showed that pituitary levels of TSHβ mRNA were higher in these animals while PVN levels of preproTRH mRNA were unchanged. These data suggest that the effects of rexinoid signaling are primarily at the level of the pituitary although a small contribution from hypothalamic TRH cannot be ruled out since these animals had higher T4 levels and no compensatory decrease preproTRH mRNA in the PVN. These data for endogenous rexinoid signaling in the mouse are further supported by our studies of the RXRγ deficient mouse which has increases in serum TSH and T4 that are comparable to this rexinoid antagonist pharmacologic model (Brown 2000
). We have also shown that RXRγ levels are similar to or slightly higher than RXRα and RXRβ levels in mouse pituitaries, and that gene deletion of RXRγ does not affect levels of the other RXR isotypes (Brown 2000
Patients with nonthyroidal illness commonly have low serum TSH and T4 levels and appear to have suppression of the HPT axis and the level of the pituitary and/or hypothalamus. Potential mechanisms include alterations in deiodinase function or cytokine signaling (Adler 2007
; Koenig 2008
). We believe that our data showing the important role of rexinoid signaling in the thyrotrope may be another feasible mechanism for the observed alterations in thyroid function in patients with nonthyroidal illness, which is a testable hypothesis.