ACTH receptor: ectopic expression, activity and signaling.

Area of Science:

• Molecular Biology
• Cell Signaling
• Endocrinology

Background:

• Ectopic expression of the adrenocorticotropic hormone receptor (ACTHR, or melanocortin 2 receptor, MC2R) in non-adrenal cells has been challenging, hindering the molecular analysis of ACTH signaling pathways.
• Mouse 3T3 fibroblasts, including Balb/c 3T3, express the MC2R accessory protein (MRAP), which is crucial for ACTHR function and proper plasma membrane insertion.
• The Y1 adrenocortical tumor cell line serves as a model for studying ACTH signaling due to its stable expression of ACTHR and functional responses to ACTH.

Purpose of the Study:

• To establish a cellular system for analyzing ACTH signaling pathways by ectopically expressing the mouse ACTHR in Balb/c mouse 3T3 fibroblasts.
• To investigate the molecular mechanisms underlying ACTH-induced gene expression, specifically the induction of fos and jun genes in these engineered cells.
• To compare the ACTH signaling pathways in engineered 3T3 fibroblasts with those in the established Y1 adrenocortical cell line.

Main Methods:

• Ectopic expression of mouse ACTHR-cDNA in Balb/c 3T3 fibroblasts using the pSVK3 mammalian plasmid vector, followed by selection for geneticin resistance and clonal isolation, creating 3T3-AR transfectants.
• Isolation of control 3T3-0 transfectants carrying the empty pSVK3 vector.
• Screening of 3T3-AR and 3T3-0 clones for response to ACTH(39), including adenylate cyclase activation and c-Fos protein induction, in comparison with Y1 cells. Signaling pathway analysis using specific inhibitors (H89/PKA, PD98059/MEK, Go6983/PKC, SP600125/JNK).

Main Results:

• Eight of fourteen 3T3-AR clones responded to ACTH(39) with adenylate cyclase activation and c-Fos induction, though levels were not strictly correlated.
• ACTH(39) induced other fos and jun genes in 3T3-AR transfectants expressing ACTHR levels similar to Y1 cells.
• In 3T3-AR transfectants, ACTH(39)-induced c-Fos was significantly inhibited by SP600125 (JNK inhibitor) and H89 (PKA inhibitor), indicating the involvement of JNK and cAMP/PKA/CREB pathways, but not MEK/ERK or PKC pathways, unlike in Y1 cells. ACTH(39) promoted JNK1/2 phosphorylation.

Conclusions:

• Ectopic expression of active ACTHR in Balb3T3 fibroblasts creates responsive cellular systems for studying ACTH signaling.
• These Balb3T3-AR cells activate cAMP/PKA/CREB and JNK pathways and induce fos and jun genes in response to ACTH.
• While these pathways are activated, they are insufficient to elicit ACTH-mediated changes in fibroblast morphology, migration, or proliferation, unlike in Y1 adrenocortical cells.