Somatotrophs are the only pituitary cells that express Ret, GFRα1 and GDNF. This study investigated the effects of Ret in a somatotroph cell line, in primary pituitary cultures and in Ret KO mice. Ret regulates somatotroph numbers by inducing Pit‐1 overexpression, leading to increased p53 expression and apoptosis, both of which can be prevented with Ret or Pit‐1 siRNA. The Pit‐1 overexpression is mediated by sustained activation of PKCδ, JNK, c/EBPα and CREB induced by a complex of Ret, caspase 3 and PKCδ. In the presence of GDNF, Akt is activated, and the Pit‐1 overexpression and resulting apoptosis are blocked. The adenopituitary of Ret KO mice is larger than normal, showing Pit‐1 and somatotroph hyperplasia. In normal animals, activation of the Ret/Pit‐1/p53 pathway by retroviral introduction of Ret blocked tumor growth in vivo. Thus, somatotrophs have an intrinsic mechanism for controlling Pit‐1/GH production through an apoptotic/survival pathway. Ret might be of value for treatment of pituitary adenomas.

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Control of polyene macrolide production in Streptomyces natalensis is mediated by the transcriptional activator PimR. This regulator combines an N-terminal domain corresponding to the Streptomyces antibiotic regulatory protein (SARP) family of transcriptional activators with a C-terminal half homologous to guanylate cyclases and large ATP-binding regulators of the LuxR family. The PimR SARP domain (PimRSARP) was expressed in Escherichia coli as a glutathione S-transferase (GST)–fused protein. Electrophoretic mobility shift assays showed that GST-PimRSARP binds a single target, the intergenic region between the regulatory genes pimR and pimMs in the pimaricin cluster. The PimRSARP-binding site was investigated by DNaseI protection studies, revealing that it contains three heptameric direct repeats adjusting to the consensus 5′-CGGCAAG-3′. Transcription start points of pimM and pimR promoters were identified by 5′-RACE, revealing that unlike other SARPs, PimRSARP does not interact with the -35 region of its target promoter. Quantitative transcriptional analysis of these regulatory genes on mutants on each of them has allowed the identification of the pimM promoter as the transcriptional target for PimR. Furthermore, the constitutive expression of pimM restored pimaricin production in a pimaricin-deficient strain carrying a deletion mutant of pimR. These results reveal that PimR exerts its positive effect on pimaricin production by controlling pimM expression level, a regulator whose gene product activates transcription from eight different promoters of pimaricin structural genes directly.

To investigate the mechanisms by which the hypothalamic peptide GHRH influences cell division, we analyzed its effects on the proliferation of two different cell lines: CHO-4, an ovary-derived cell line, and GH3, a pituitary-derived cell line. We found that GHRH induces the proliferation of pituitary-derived cells but inhibits the proliferation of ovary-derived cells. We further characterized this dual effect of GHRH to find that the cytoplasmic signals induced by this hormone are similar in both cell lines. Moreover, in CHO-4 cells GHRH stimulates two well-known positive cell cycle regulators, c-myc and cyclin D1, but is unable to induce the degradation of the negative cell cycle regulator p27(Kip1). Significantly, when the Pit-1/GHF-1 gene is exogenously expressed in CHO-4 cells, the negative effect of GHRH on the proliferation of these cells is attenuated. Furthermore, when the levels of Pit-1 are downregulated by siRNA in GH3-GHRHR cells, the positive effects of GHRH on the proliferation of these cells are diminished. These findings add to our understanding of the molecules involved in the regulation of cell proliferation by GHRH, as we demonstrate for the first time that Pit-1 is not only required to drive the expression of the GHRH receptor, as previously described, but is also needed for the downstream effects that occur after its activation to modulate cell proliferation. These data suggest that the regulation of cell proliferation in response to a specific growth factor depends in certain cell populations on the presence of a tissue-specific transcription factor.