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Abstract: Failure in obtaining expression of functional adrenocorticotropic hormone receptor (ACTHR, or melanocortin 2 receptor, MC2R) in non-adrenal cells has hindered molecular analysis of ACTH signaling pathways. Here, we ectopically expressed the mouse ACTHR in Balb/c mouse 3T3 fibroblasts to analyze ACTH signaling pathways involved in induction of fos and jun genes. Natural constitutive expression of the MC2R accessory protein (MRAP) in Balb3T3 and other mouse 3T3 fibroblasts (NIH, Swiss and 3T3-L1) renders these fibroblastic lines suitable for ectopic expression of ACTHR in its active form properly inserted into the plasma membrane at levels similar to those found in mouse Y1 adrenocortical tumor cells. The Y1 cell line is a cultured cell system well known for stably displaying normal adrenal specific metabolic pathways, ACTHR expression and ACTH functional responses. Thirty-nine sub-lines expressing ACTHR (3T3-AR transfectants) were selected for geneticin-resistance and clonally isolated after transfection of ACTHR-cDNA (in the pSVK3 mammalian plasmidial vector) into Balb3T3 fibroblasts. In addition, sixteen clonal sub-lines of Balb3T3 (3T3-0 transfectants) carrying the pSVK3 empty vector were likewise isolated. Fourteen 3T3-AR and four 3T3-0 clones were screened for response to ACTH39 in comparison with Y1 adrenocortical cells. Eight 3T3-AR clones responded to ACTH39 with activation of adenylate cyclase and induction of c-Fos protein, but the levels of, respectively, activation and induction were not strictly correlated. Other fos and jun genes were also induced by ACTH39 in 3T3-AR transfectants, which express levels of ACTHR protein similar to parental Y1 cells. Signaling pathways relevant to c-Fos induction was extensively investigated in 3 clones: 3T3-AR01 and –07 and 3T3-04. In Y1 cells, specific inhibitors (H89/PKA; PD98059/MEK; Go6983/PKC and SP600125/JNK) show that signals initiated in the ACTH/ACTHR-system activate 4 pathways to induce the c-fos gene, namely: (a) cAMP/PKA/CREB; (b) MEK/ERK1/2; (c) PKC and d) JNK1/2. In 3T3-AR transfectants, both inhibitors PD98059 and Go6983 proved completely ineffective to inhibit c-Fos induction by ACTH39, implying that MEK/ERK and PKC pathways are not involved in this process. On the other hand, SP600125 caused 85% inhibition of c-Fos induction by ACTH39 and, in addition, ACTH39 promotes JNK1/2 phosphorylation, suggesting that JNK is a major signaling pathway mediating c-Fos induction by ACTH39 in these cells. In addiction, PKA inhibitor H89 also inhibits c-Fos induction in 3T3-AR7 cells by ACTH39, implicating activation of the cAMP/PKA/CREB pathway in c-Fos induction by ACTH39. However, the cAMP derivatives db-cAMP and 8Br-cAMP, do not promote CREB phosphorylation and c-Fos induction in parental Balb3T3 and 3T3-AR transfectants, confirming previous report by others. In conclusion, expression of active ACTHR in Balb3T3 fibroblasts renders these cells responsive to ACTH with activation of cAMP/PKA/CREB and JNK pathways and, also, induction of genes from the fos and jun families. These results show that Balb 3T3-AR sublines are useful cellular systems for genetic analysis of ACTH-signaling pathways. However, activation of cAMP/PKA/CREB and JNK pathways and induction of fos and jun genes are not yet sufficient to enable ACTH for interference in morphology, migration and proliferation of Balb3T3 fibroblasts as it does in Y1 adrenocortical cells.

ACTH is the hormone known to control adrenal cortex function and maintenance in the intact animal but, in culture, it inhibits proliferation of adrenocortical cells from different mammalian species, a puzzle that has remained unsolved for nearly 30 years. In this paper we compare ACTH and fibroblast growth factor 2 (FGF2) antagonistic effects on the cell cycle in the Y1 cell line, a functional lineage of mouse adreno-cortical tumor cells. This cell line displays chronic high levels of c-Ki-Ras-GTP, high active constitutive levels of phosphatidylinositol 3-OH kinase/Protein Kinase B (PI3K/AKT) and low constitutive basal expression of c-Myc, which accounts for a minor deregulation of the cell cycle. In G0/G1-arrested Y1 cells, over-expression of the dominant negative mutant HaRasN17 drastically reduces c-Ki-Ras-GTP levels, eliminating basal c-Myc expression and basal S phase entry. PI3K/Akt seems to be the downstream pathway from c-Ki-ras for deregulation of c-Myc basal expression, since wortmannin abolishes c-Myc expression in serum-starved, G0/G1-arrested Y1 cells. FGF2 is a strong mitogen for Y1 cells, promoting – in a manner dependent on the MEK/ERK pathway – c-myc transcription induction, c-Myc protein stabilization and S phase entry in G0/G1-arrested Y1 cells. On the other hand, ACTH causes c-Myc protein destabilization, partially blocking S phase entry induced by FGF2, by a process dependent on the cAMP/protein kinase A (PKA) pathway. The whole pathway activated by ACTH to destabilize c-Myc protein in Y1 cells might comprise the following steps: ACTH receptor →cAMP/PKA → Akt deactivation →GSK3 activity liberation → c-Myc Thr58 phosphorylation.We demonstrate that c-Myc regulation is a central key in the cell cycle control by these factors, since enforced expression of c-Myc through the MycER chimera abrogates the ACTH inhibitory effect over FGF2-induced S phase entry.

3T3 CELLS are embryonic mouse fibroblasts established in culture1. Serum depletion or “step-down” causes them to enter a “resting” state, which they leave on restoration of serum or “step-up”, entering the “proliferative” state2–4. This transition between resting (also called G0, A or R) and proliferating states seems to be the critical step in cell growth control5–7. Its study has been aided recently by several observations on extracellular growth regulators for 3T3 cells, for example the discovery of a new pituitary growth factor4,8, growth stimulation by glucocorticoids4,9and interactions between pituitary factor and classical hormones10–12. Further studies would be greatly helped if variants or mutants of 3T3 with different growth responses towards hormones could be isolated. Here we report our finding of the variant ST1 which is inhibited by glucocorticoids, unlike 3T3 cells whose growth is stimulated by glucocorticoids4,9(growth of fibroblasts is normally inhibited by glucocorticoids13). In this paper we discuss the value of this new cell type for studies of the resting–proliferative transition; elsewhere we shall report how the adhesion of this variant to the substrate is greatly affected by both the removal of serum and the addition of cyclic AMP.

We previously reported that ACTH, but not dibutyryl cAMP, rapidly induces the c-fos proto-oncogene in Y-1 adrenocortical cells.

The effects of Mg2+ and Ca2+ deprivation on survival and growth of normal and transformed cultured cells were studied. Normal fibroblast from several origins (mouse, hamster and human) did not grow in 2 µM Mg2+ medium, whereas transformed fibroblasts (mouse and rat) and tumor cells of other types (mouse adrenocortical and rat glial cells) grew optimal. All transformed or tumorogenic cells that showed low growth requirement for Mg2+ were able to develop colonies in agarose suspension cultures, i.e., were anchorage-independent for growth. Mg2+ deprivation of normal cells did not lead to cell cycle arresting at Go/ Gl and cell death accounts for the limited growth of these cells in medium containing low Mg2+ concentration. Contrary to Mg2+, starvation for serum or for Ca2+ caused normal cells to undergo cell cycle arrest at the Go/Gl phase. During the progressive spontaneous transformation of Swiss mouse 3T3 fibroblasts, the decrease in growth requirements for Mg2+ and Ca2+ do not occur at the same time. The requirement for external Ca2+ lowers before the onset of anchorage-independent growth while the Mg2+ requirement only decreases after cells become anchorage-independent. Therefore the reductions in growth requirement for Mg2+ and Ca2+ that take place in cell transformation are not linked events.

The adrenocorticotropic hormone (ACTH) inhibits the growth of Y1 mouse adrenocortical tumor cells as well as normal adrenocortical cells in culture but stimulates adrenocortical cell growth in vivo. In this study, we investigated this paradoxical effect of ACTH on cell proliferation in Y1 adrenal cells and have unmasked a growth-promoting effect of the hormone. Y1 cells were arrested in the G1phase of the cell cycle by serum starvation and monitored for progression through S phase by measuring [3H]thymidine incorporation into DNA and by measuring the number of nuclei labeled with bromodeoxyuridine. Y1 cells were stimulated to progress through S phase and to divide after a brief pulse of ACTH (up to 2 h). This effect of ACTH appeared to be cAMP independent, since ACTH also induced cell cycle progression in Kin-8, a Y1 mutant with defective cAMP-dependent protein kinase activity. The growth-promoting effect of ACTH in Y1 was preceded by the rapid activation of p44 and p42 mitogen-activated protein kinases and by the accumulation of c-FOS protein. In contrast, continuous treatment with ACTH (14 h) inhibited cell cycle progression in Y1 cells by a cAMP-dependent pathway. The inhibitory effect of ACTH mapped to the midpoint of G1. Together, the results demonstrate a dual effect of ACTH on cell cycle progress, a cAMP-independent growth-promoting effect early in G1possibly mediated by mitogen-activated protein kinase and c-FOS, and a cAMP-dependent inhibitory effect at mid-G1. It is suggested that the growth-inhibitory effect of ACTH at mid-G1represents an ACTH-regulated check point that limits cell cycle progression.

Results are reported showing that pituitary extracts stimulate growth of 3T3 cells, an established line of mouse fibroblast. Cells were cultured in medium containing an organic buffer (HEPES; N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid), which increases the serum requirement for cell growth. The pituitary growth-prompting activity is (a) not dialyzable; (b) thermolabile; (c) protease sensitive; and (d) enhanced by hydrocortisone (a steroid hormone). Activity was quantitatively assayed by measurement of stimulation of cell division or [3H]thymidine uptake into DNA. The active species is probably different from pituitary protein hormones and it seems to be effective at concentrations observed for protein hormones in vivo. 3T3 cells transformed by simian virus 40 are not responsive to this activity.