Transcriptional regulation of the thyrotropin-releasing hormone gene by leptin and melanocortin signaling

Abstract

Starvation causes a rapid reduction in thyroid hormone levels in rodents. This adaptive response is caused by a reduction in thyrotropin-releasing hormone (TRH) expression that can be reversed by the administration of leptin. Here we examined hypothalamic signaling pathways engaged by leptin to upregulate TRH gene expression. As assessed by leptin-induced expression of suppressor of cytokine signaling-3 (SOCS-3) in fasted rats, TRH neurons in the paraventricular nucleus are activated directly by leptin. To a greater degree, they also contain melanocortin-4 receptors (MC4Rs), implying that leptin can act directly or indirectly by increasing the production of the MC4R ligand, alpha-melanocyte stimulating hormone (alpha-MSH), to regulate TRH expression. We further demonstrate that both pathways converge on the TRH promoter. The melanocortin system activates the TRH promoter through the phosphorylation and DNA binding of the cAMP response element binding protein (CREB), and leptin signaling directly regulates the TRH promoter through the phosphorylation of signal transducer and activator of transcription 3 (Stat3). Indeed, a novel Stat-response element in the TRH promoter is necessary for leptin's effect. Thus, the TRH promoter is an ideal target for further characterizing the integration of transcriptional pathways through which leptin acts.

Figure 1

TRH neurons in the rat PVH express the MC4R and SOCS-3 after leptin administration. (a and b) Double label in situ hybridization was performed for the MC4R and TRH on hypothalamic sections. Shown is a caudal medial parvocellular section. TRH neurons are darkly stained (TRH-dig), whereas the granules represent MC4R expression (MC4R-³⁵S). In this image, all the TRH neurons coexpress MC4R. (c and d) A similar study was performed to colocalize SOCS-3 (SOCS-3-³⁵S) expression after saline or leptin treatment in rats fasted for 48 hours. The arrows indicate the double-labeled neurons.

Figure 2

Leptin and α-MSH signaling pathways activate the hTRH promoter. (a) 293T cells were transfected with both the MC4R and ObRb and a hTRH luciferase construct. Cells were stimulated with 100 nM of the indicated ligand. The fold stimulation is shown over each treatment group. (b) Nuclear extract from 293T cells transfected in duplicate was prepared 15 minutes after stimulation with α-MSH. Equal amounts of protein from control and treated cells were probed using phospho-CREB and CREB antibodies. The duplicate results are shown. (c) Nuclear extracts were prepared from transfected 293T cells treated with leptin at the indicated times and used in EMSA with the radiolabeled SIEm67 element. Complexes were identified by using specific antibodies against Stat isoforms (αS1, against Stat1; αS3, against Stat3). The antibodies were used on extract stimulated for 15 minutes.

Figure 3

A near consensus cAMP response element mediates the α-MSH response via CREB. (a) The cis-acting sequence mediating the response to α-MSH was mapped in 293T cells using the indicated hTRH promoter complexes. (b) A dominant CREB mutant (S133A) or empty vector (PKCR2) was cotransfected in the presence of the –900 TRH reporter construct, and the effects on stimulation were examined after the addition of α-MSH. The data are expressed as fold stimulation. (c) The binding of CREB to Site 4 was assessed using EMSA. Control (NE) or CREB-transfected (C-NE) nuclear extract was incubated with a radiolabeled Site 4 probe. The specificity of CREB binding is demonstrated by its supershift with anti-CREB (αC) antiserum.

Figure 4

T3 blocks α-MSH–mediated upregulation of the hTRH promoter. (a) 293T cells were cotransfected with expression vectors for the TRβ isoforms. The cells were incubated with either 100 nM α-MSH alone or with α-MSH and 100 nM T3. The data are expressed as fold stimulation from untreated cells. (b) EMSA was performed with radiolabeled Site 4 and CREB-transfected nuclear extract in the presence or absence of in vitro translated hTRβ2. (c) GST-TRβ1 or GST-TRβ2 was incubated with radiolabeled SRC-1 or CREB either in the presence or absence of 1 μM T3. Lane 9, CREB (C) input. Lane 10, SRC-1 (S) input.

Figure 5

Leptin responsiveness maps to a specific region of the hTRH promoter. (a) 293T cells were transfected with a variety of hTRH reporter constructs. Their response to 100 nM leptin was then determined. The data are expressed as relative luciferase activity, and the fold response is indicated above each construct. (b) The promoter sequences of the human, rat, and murine TRH genes are compared in the area of Stat responsiveness. The Stat-responsive element is boldface and located between –141 and –132. The SP-1 site is boldface and underlined and located between –125 and –119. Also shown are the mutations made to create the Statm and ΔSP-1 constructs. (c) 293T cells were transfected with the mTRH reporter construct in an identical paradigm, and its response to leptin was assessed. nTRE, negative thyroid hormone response element.

Figure 6

Identification of the Stat binding site in the TRH promoter. (a) 293T cells were cotransfected with the indicated hTRH constructs. Leptin responsiveness was then determined. The data are expressed as percent of maximal response where the fold stimulation of the –150 construct is set at 100%. (b) EMSA was performed with a radiolabeled probe spanning the human Stat site and nuclear extract from leptin-treated or untreated cells. The specificity of the bound complex was discerned by using an anti-Flag (αF) antibody. (c) A similar EMSA paradigm was used on the canonical SIEm67 probe. (d) A similar EMSA paradigm was used on a probe spanning the murine Stat element. (d) EMSA was performed on the murine Stat element with the addition of cold oligonucleotides representing the murine (lanes 3 and 4) or human (lanes 5 and 6) Stat elements. A 10× cold competitor was added in lanes 3 and 5, and 100× in lanes 4 and 6.

Figure 7

The effect of Stat isoforms on TRH promoter activity. (a) 293T cells were cotransfected with the indicated TRH constructs or Gas-Luc with ObRb in the presence or absence of Stat1. The effect of Stat1 is quantified as fold enhancement over values from cells that did not receive Stat1. (b) EMSA was performed with the canonical SIEm67 probe and NE from Stat1 or Stat3 transfected cells. The indicated complexes are shown.

Figure 8

A model of leptin action on the TRH neuron. Shown is a proposed model of leptin action on the TRH neuron. After crossing the blood-brain barrier leptin can act either directly on the TRH neuron or via an indirect pathway by increasing the production of α-MSH in the POMC neuron in the arcuate nucleus. Also shown are the inputs of AGRP and NPY, both of which are known to project to the TRH neuron. Leptin, through the phosphorylation of Stat3, can directly activate the TRH promoter as can α-MSH through the activation of CREB. T3 can inhibit TRH gene expression, principally through the TRβ2 isoform. NPYR1/5, NPY receptor 1 or 5.