Nescient Helix-Loop-Helix 2 Interacts with Signal Transducer and Activator of Transcription 3 to Regulate Transcription of Prohormone Convertase 1/3

Abstract

Mechanisms controlling body weight involve gene regulation through the activation of signal transduction pathways. The Janus kinase/signal transducer and activator of transcription (STAT) signal transduction pathway is the mechanism primarily used by leptin in the hypothalamus. The transcription factor nescient helix-loop-helix 2 (Nhlh2) is a downstream target of leptin signaling and is expressed in proopiomelanocortin arcuate neurons. Proopiomelanocortin is cleaved by prohormone convertase 1/3 (PC1/3) to produce peptides that regulate the body's response to energy availability. Previous studies show that the PC1/3 promoter contains STAT3 sites mediating leptin-induced PC1/3 expression, and that Nhlh2 is required for hypothalamic PC1/3 expression because Nhlh2 knockout mice have reduced PC1/3 mRNA levels. Studies herein reveal that leptin-induced PC1/3 gene expression is abrogated in N2KO mice, and that in a hypothalamic cell line both STAT3 and Nhlh2 are required for the full transcriptional response of a PC1/3 reporter gene after leptin stimulation. Furthermore, it is shown that Nhlh2 binds to E-box motifs found adjacent to STAT3 sites in the PC1/3 promoter both in vitro and in chromatin immunoprecipitation assays. Finally, two different protein-protein interaction assays confirm the presence of a STAT3:Nhlh2 heterodimer on the PC1/3 promoter. The Nhlh2:STAT3 heterodimer may be an important transcriptional regulator of other hypothalamic genes in the leptin signaling pathway. These data confirm Nhlh2 as an integral element of the Janus kinase/STAT signaling pathway and are the first to demonstrate coordinated control of PC1/3 transcription by Nhlh2 and STAT3 after leptin stimulation.

Stromal cell-derived factor-1 (SDF-1)/chemokine (C-X-C motif) receptor 4 (CXCR4) axis activation induces intra-islet glucagon-like peptide-1 (GLP-1) production and enhances beta cell survival

“…4b). These findings were of interest because Akt and JAK/STAT signalling are known to activate the expression of Pc1/3 [23, 24]. PC1/3 is required for the processing of proglucagon, the product of the glucagon gene ( Gcg ), to GLP-1 [25].…”

Leptin signaling regulates hypothalamic expression of nescient helix-loop-helix 2 (Nhlh2) through signal transducer and activator 3 (Stat3)

“…As we have previously identified key transcriptional targets for Nhlh2 (PC1/3 and Mc4r, for example (Fox and Good, 2008, Wankhade and Good, 2011)), these studies allow us to begin to piece together the molecular pathways between peripheral increases in leptin and hypothalamic control responses. Obesity can result in both high leptin levels and high levels of inflammatory cytokines such as IL6 (Cancello et al, 2004).…”

“…These results identify another Stat3-regulated gene, and as Nhlh2 is itself a transcription factor, identify a mechanism for induction of many possible Nhlh2-regulated target genes. The PC1/3 gene, for example, requires both Nhlh2 and Stat3 to coordinately control its leptin-induced expression (Fox and Good, 2008). Mechanistically, with these results, leptin is now shown to stimulate hypothalamic Stat3, and within the ARC, this leads to transcriptional regulation of POMC, Nhlh2 and, together with newly synthesized Nhlh2, the enzyme needed to process POMC, PC1/3.…”

“…Large-scale plasmid preparation was performed using a Qiagen Midi Plasmid kit (Qiagen Ltd., Valencia, CA). The plasmid construct containing Stat3 was a generous gift from Dr. James Darnell, The Rockefeller University, New York, NY, and was modified to have an improved translational start site (Fox and Good, 2008). The leptin receptor (ObRB) plasmid was a generous gift from, Christian Bjørbæk, Harvard Medical School, Boston, MA.…”

Loss of NSCL-2 in Gonadotropin Releasing Hormone Neurons Leads to Reduction of Pro-Opiomelanocortin Neurons in Specific Hypothalamic Nuclei and Causes Visceral Obesity

“…So far, it was not clear whether loss of NSCL-2 expression in GnRH neurons is responsible for reduction of GnRH neurons and reduced fertility of Nscl-2 mutants, although it has been demonstrated that NSCL-2 and its close relative, NSCL-1, are concomitantly involved in the control of differentiation and migration of GnRH neurons probably via regulation of necdin expression (Krüger et al, 2004). In response to leptin signaling, NSCL-2 regulates expression of the prohormone convertase 1/3 (PC1/3) in hypothalamic neurons (Fox and Good, 2008), which is required for proteolytic processing of pro-opiomelanocortin (POMC) into ACTH and ␣-melanocytestimulating hormone (␣-MSH). ␣-MSH restricts food intake by signaling via melanocortin-4 receptor (MC4-R) and mediates increased energy expenditure (Elmquist, 2001).…”