GHRP-6 increases growth hormone by reducing miR-709, an inhibitor that targets the PKCα pathway in pituitary cells

Pituitary growth hormone (GH) is a cornerstone of organismal growth and metabolic regulation. While peptide secretagogues like GHRP-6 are known to stimulate its release, the intricate intracellular mechanisms, particularly the role of non-coding RNAs, are still being unraveled. MicroRNAs (miRNAs) are small RNA molecules that can fine-tune gene expression and participate in diverse biological processes. A significant gap exists in understanding how miRNAs regulate pituitary GH synthesis during growth phases. This study investigates miR-709, the second most highly expressed miRNA in the pituitary, to clarify its potential role as a negative regulator of GH production and to explore how compounds like GHRP-6 might interact with this regulatory layer. The central question is whether miR-709 directly controls key signaling components like Protein Kinase C alpha (PKCα), encoded by the PRKCA gene, to modulate GH output.

This study used a combination of in vitro and in vivo models to dissect the regulatory pathway. Researchers utilized the rat pituitary tumor cell line GH3 for their in vitro experiments. They employed bioinformatics analysis and a dual luciferase reporter assay to confirm that PRKCA is a direct target of miR-709. To test the functional relationship, they over-expressed miR-709 in GH3 cells and measured changes in Prkca transcription and translation levels, as well as the activity of downstream signaling pathways (ERK1/2, cAMP/PKA) and transcription factors (CREB1) using western blot. Finally, they treated GH3 cells and live animal models with GHRP-6 to observe its effect on miR-709, PKCα, and GH expression.

Over-expression of miR-709 significantly inhibited GH synthesis and suppressed the viability of GH3 pituitary cells. This inhibitory effect was traced to its direct targeting of the PRKCA gene, which codes for PKCα. Increased miR-709 levels led to a marked reduction in both Prkca transcription and translation. This suppression of PKCα subsequently inhibited the ERK1/2 signaling pathway and the transcription factor CREB1, a key regulator of the Gh1 gene. The researchers also noted potential crosstalk with the cAMP/PKA signaling pathway.

Conversely, treatment with GHRP-6 in both in vitro and in vivo models increased PKCα and GH expression while simultaneously reducing miR-709 expression.

This finding was further solidified when GHRP-6 was shown to attenuate the inhibitory effect that miR-709 normally exerts on GH expression, confirming an antagonistic relationship between the peptide and the microRNA in the regulation of growth hormone synthesis.