Activation of α1A-adrenergic receptor promotes differentiation of rat-1 fibroblasts to a smooth muscle-like phenotype

Abstract

BackgroundFibroblasts, as connective tissue cells, are able to transform into another cell type including smooth muscle cells. α1A-adrenergic receptor (α1A-AR) stimulation in rat-1 fibroblasts is coupled to cAMP production. However, the significance of an increase in cAMP produced by α1A-AR stimulation on proliferation, hypertrophy and differentiation in these cells is not known.ResultsActivation of the α1A-AR in rat-1 fibroblasts by phenylephrine (PE) inhibited DNA synthesis by 67% and blocked the re-entry of 81% of the cells into S phase of the cell cycle. This cell cycle blockage was associated with hypertrophy characterized by an increase in protein synthesis (64%) and cell size. Elevation of cAMP levels decreased both DNA and protein synthesis. Inhibition of adenylyl cyclase or protein kinase A reversed the antiproliferative effect of cAMP analogs but not PE; the hypertrophic effect of PE was also not altered. The functional response of rat-1 cells to PE was accompanied by increased expression of cyclin-dependent kinase (Cdk) inhibitors p27kip1 and p21cip1/waf1, which function as negative regulators of the cell cycle. Stimulation of α1A-AR also upregulated the cell cycle regulatory proteins pRb, cyclin D1, Cdk 2, Cdk 4, and proliferating cell nuclear antigen. The antiproliferative effect of PE was blocked by p27kip1 antisense but not sense oligonucleotide. PE also promoted expression of smooth muscle cell differentiation markers (smooth muscle alpha actin, caldesmon, and myosin heavy chain) as well as the muscle development marker MyoD.ConclusionsStimulation of α1A-AR promotes cell cycle arrest, hypertrophy and differentiation of rat-1 fibroblasts into smooth muscle-like cells and expression of negative cell cycle regulators by a mechanism independent of the cAMP/PKA signaling pathway.

Stimulation of α1a Adrenergic Receptors Induces Cellular Proliferation or Antiproliferative Hypertrophy Dependent Solely on Agonist Concentration

“…In these experiments, rat-1 cells stably expressing HA-α 1a AR were incubated for 24, 48 and 72 hours with various concentrations of PE (10 −10 to 10 −5 M) and the effects of agonist stimulation on proliferative and hypertrophic growth responses determined by side-by-side cell counts, total cellular protein measurement and morphological observation. As previously reported [38], [39], high doses of PE (10 −6 to 10 −5 M) are strongly antiproliferative and induced a visually evident decrease in cell number (Fig. 1A) quantitatively established by cell counting (Fig.…”

“…This variability is consistent with contradictory α 1a AR signaling responses that are not easily distinguished from responses of the other subtypes. Consistent with phenotypic results in expression models [38], [39], pharmacologically dissection in primary foreskin fibroblasts suggests the α 1b AR mediates proliferation rather than the equally expressed α 1a AR. [67].…”

“…Generally, α 1a AR stimulation has been associated with antiproliferative [38] and [39], [65] hypertrophic phenotypes; however, this receptors ability to induce proliferative [46] and protective [66] responses demonstrates a diversity of biological functions. This variability is consistent with contradictory α 1a AR signaling responses that are not easily distinguished from responses of the other subtypes.…”

“…In these models, comparison of signaling efficacy between individual subtypes has shown α 1a AR signaling to be more robust in HeLa [10], rat-1 fibroblast [22], [34], [35], HEK293 [34], SK-N-MC (1996theroux) and CHO [36] cells, although the relationship between canonical signaling intensity and α 1 AR-induced phenotypic responses [36]–[39] remains unclear. Beyond signaling intensity, there are subtype specific mechanisms such as the rapid internalization [40] and proliferative phenotypes [38] of the α 1b AR that contrast with the slow internalization [41] and antiproliferative [38] phenotypes α 1a AR.…”

Long-term α1B-adrenergic receptor activation shortens lifespan, while α1A-adrenergic receptor stimulation prolongs lifespan in association with decreased cancer incidence

“…In most cell types studied, the α 1A AR subtype increases the levels of the cdk inhibitor p27Kip1 and halts the cell cycle at the G1-S checkpoint (Shibata et al 2003;Saeed et al 2004;Gonzalez-Cabrera et al 2004). In our study, there was a significant reduction in cancer incidence in CAM-α 1A AR mice, which could be due to this cell cycle stoppage.…”