Brief treatments with forskolin enhance s-phase entry in balance epithelia from the ears of rats

Abstract

In the ears of mammals, hair cell loss results in permanent hearing and balance deficits, whereas in fish, amphibians, and birds, the production of replacement hair cells can restore those modalities. In avian ears, continuous exposures to forskolin trigger cell proliferation and the regeneration of hair cells, so we investigated the effect of forskolin on sensory epithelia cultured from the ears of mammals. Continuous 72 hr exposures to forskolin failed to induce proliferation in neonatal rat utricles, but brief (</=1 hr) exposures to forskolin or Br-cAMP did. Proliferation occurred only in media that contained serum. Forskolin also augmented the mitogenic effects of glial growth factor 2. The S-phase entry induced by forskolin was blocked by monensin and bafilomycin, two compounds that can inhibit the recycling of membrane receptors. The results are consistent with the hypothesis that in mammalian vestibular epithelia elevated cAMP induces S-phase entry by increasing the number of growth factor receptors at the plasma membrane.

Fig. 1.

Continuous exposure to 0.1–100 μmforskolin does not stimulate S-phase entry in mammalian utricle cells.A, B, Pieces of utricular sensory epithelium fixed after 72 hr in culture in the standard medium in the continuous presence of 10 μm (A) or 100 μm(B) forskolin (Forsk). Nuclei of cells that entered S-phase and incorporated BrdU were stainedblack after immunocytochemistry and were visualized by differential interference contrast (DIC) microscopy. C, Histogram of the level of BrdU labeling in epithelia showing no significant increase in S-phase entry after a 72 hr treatment with forskolin when compared with control cultures in 1% DMSO.Inset, Timeline. The standard medium consisted of 2.5% FBS and 3 μg/ml BrdU with either 1% DMSO (Control) or forskolin at 0.1–100 μm present from the start of the experiment (S). After 72 hr in culture the tissues were fixed (F) and processed for immunocytochemistry. Scale bars, 100 μm.

Fig. 2.

Continuous exposures to forskolin at 0.1 and 1 μm enhanced rhGGF2-mediated induction of S-phase entry, whereas exposures to 10 and 100 μm forskolin reduced the induction of S-phase that was evoked by rhGGF2. A, Timeline is shown. Pieces of epithelium were cultured for 72 hr in a standard medium containing either 50 ng/ml rhGGF2 (GGF2) or forskolin (Forsk) at concentrations ranging from 0.1 to 100 μm. B, Forskolin (1 μm) potentiated the rhGGF2 effect, whereas at higher concentrations (10 and 100 μm), forskolin significantly inhibited the rhGGF2-induced S-phase entry. *, Significant difference from cultures treated with GGF2(p < 0.05). F, Fixed;S, start.

Fig. 3.

Continuous exposure to Br-cAMP significantly inhibits rhGGF2-induced S-phase entry. A, Timeline is shown. Pieces of epithelium were cultured for 72 hr in a standard medium containing either rhGGF2 alone (GGF2) or Br-cAMP from 0.1 to 1 mm in the presence of rhGGF2.B, At 0.1, 0.5, or 1 mm, the Br-cAMP significantly inhibited the rhGGF2-induced S-phase entry. *, Significant difference when compared with GGF2(p < 0.001). F, Fixed;S, start.

Fig. 4.

Brief treatments with forskolin or Br-cAMP induce 11- to 12-fold increases in S-phase entry. A, Timeline is shown. Pieces of epithelium were treated for 15 min with 1 μm forskolin (Forsk) or for 1 hr with 0.5 mm Br-cAMP in a standard medium. The forskolin- and Br-cAMP-containing media were then replaced with a standard medium for the remainder of the 72 hr culture period. In this and all following figures, discontinuity indicators show that the timelines are schematic and not to scale. B, C, When pieces of utricles were treated briefly with forskolin (Forsk 15 min), the level of S-phase entry was ∼12-fold higher than that in control epithelia. When other pieces of epithelium were treated for 1 hr with Br-cAMP (Br-cAMP 1 hr), the level of S-phase entry was ∼11-fold higher than that in the control epithelia. *, Significant difference when compared with Control(p < 0.05). Scale bars, 100 μm.F, Fixed; S, scale.

Fig. 5.

Treatment with a PKA inhibitor suggests dependence on PKA for the increase in S-phase induced by brief treatment with forskolin. A, B, Timelines are shown. A, Pieces of epithelium were incubated for 15 min with 1 μmforskolin (Forsk) and then maintained in a standard medium for the remainder of the 72 hr culture period. Other pieces of utricle were preincubated for 60 min with 0.5, 1, or 5 μmKT5720 in a medium containing 2.5% FBS but no BrdU. The medium was then changed for a standard medium containing the inhibitor and 1 μm forskolin for 15 min. The forskolin was removed, and the pieces of epithelium were maintained in a standard medium containing KT5720 for 72 hr. B, Pieces of epithelium were treated for 1 hr with 0.5 mm Br-cAMP in a standard medium. The Br-cAMP-containing medium was then replaced with a standard medium for the remainder of the 72 hr culture period. Other pieces of epithelium were preincubated for 60 min with 5 μm KT5720 in a medium containing 2.5% FBS but no BrdU. The medium was then changed for a standard medium containing the inhibitor and 0.5 mm Br-cAMP for 1 hr. The Br-cAMP was removed, and the pieces of epithelium were maintained in a standard medium containing 5 μm KT5720 for 72 hr. C, Exposure to KT5720 induced a dose-dependent inhibition, reducing the forskolin-induced effect by 52% at 1 μm KT5720 and by 92.4% at 5 μm KT5720. Treatment with 5 μm KT5720 reduced the short-term Br-cAMP effect by 85.2% *, Significant difference when compared with Forsk 15 min(p < 0.05). **, Significant difference when compared with Br-cAMP 1 hr (p< 0.001). F, Fixed; S, start.

Fig. 6.

A 15 min treatment with 1 μmforskolin more than doubles the magnitude of S-phase response induced by rhGGF2. A, Timeline is shown. Pieces of epithelium were incubated for 15 min with 1 μm forskolin (Forsk) in a standard medium. The medium was then replaced by a medium containing rhGGF2 with BrdU and FBS for the remainder of the 72 hr culture period. B, DIC micrographs show many black BrdU-labeled nuclei in a piece of utricular epithelium treated with rhGGF2 (GGF2) and the strong increase induced by the brief treatment with forskolin (Forsk 15 min + GGF2). C, The brief treatment with forskolin increased the rhGGF2-induced entry in S-phase up to 52%. *, Significant difference when compared withGGF2 (p < 0.001). Scale bars, 100 μm. F, Fixed; S, start.

Fig. 7.

Receptor recycling prevents S-phase induction triggered by brief treatment with forskolin. A, B, Timelines are shown. A, Pieces of epithelium were preincubated for 75 min (30/15/30 min) with 0.5 μm monensin (Mon) or 0.1 μmbafilomycin A1 (Baf). During the first 30 min, the medium did not contain BrdU. For the next 45 min, the medium contained FBS and BrdU. The pieces were then maintained in an FBS-containing medium with BrdU for the remainder of the 72 hr culture.B, Other pieces were pretreated with 0.5 μm monensin (Mon) or 0.1 μmbafilomycin A1 (Baf) for 30 min in a medium devoid of BrdU. The medium was then changed for a standard medium containing 1 μm forskolin (Forsk) for 15 min and replaced by a medium containing 0.5 μm monensin or 0.1 μm bafilomycin A1 and FBS and BrdU for 30 additional minutes. Finally, the monensin or the bafilomycin A1 was removed, and the pieces of utricle were cultured in a standard medium for the remainder of the 72 hr culture period. C, The 75 min (30/15/30 min) treatment with 0.5 μmmonensin or 0.1 μm bafilomycin A1 reduced the level of S-phase entry as compared with the control, but this was not significant. Treatment with monensin inhibited the 15 min forskolin effect up to 99% (Forsk 15 min + Mon), whereas treatment with bafilomycin A1 induced a 70.5% inhibition (Forsk 15 min + Baf). * Significant difference when compared with Forsk 15 min (p < 0.001). F, Fixed; S, start.

Fig. 8.

Monensin treatment inhibits the rhGGF2-induced S-phase entry by half. A, B, Timelines are shown.A, Pieces of epithelia were cultured in a standard medium containing 50 ng/ml rhGGF2 (GGF2) for 72 hr.B, Twenty pieces were pretreated with 0.5 μm monensin (Mon) for 30 min in a medium containing FBS but no BrdU. The medium was then changed for a fresh medium containing FBS and BrdU for 45 min. Then, the medium was replaced by a medium containing rhGGF2, FBS, and BrdU for the remainder of the 72 hr culture period. C, Treatment with monensin resulted in a significant reduction of the rhGGF2-induced effect, but we were still able to see ∼50% of the effect triggered by rhGGF2 alone. *, Significant difference when compared with GGF2(p < 0.05). F, Fixed;S, start.

Fig. 9.

Absence of serum prevents S-phase induction triggered by brief treatment with forskolin. A, B, Timelines are shown. Pieces of epithelium were treated for 15 min with 1 μm forskolin (Forsk) in a serum-containing medium (A) or in a defined medium (B) composed of DMEM/F12 supplemented with transferrin, sodium selenite, hydrocortisone, and insulin in the presence of BrdU. The medium was then changed for fresh medium containing BrdU for the reminder of the 72 hr culture period.C, The level of forskolin-induced S-phase entry was strongly reduced in the defined medium (Forsk 15 min defined) when compared with the standard medium (Forsk 15 min 2.5% FBS). *, Significant difference when compared withForsk 15 min 2.5% FBS (p < 0.05). F, Fixed; S, start.

Fig. 10.

A working model of forskolin regulation of S-phase entry in mammalian supporting cells. An increase in intracellular cAMP is achieved either via forskolin activating the AC or via Br-cAMP crossing the plasma membrane. Prolonged treatment (72 hr) with forskolin or Br-cAMP alone did not induce stimulation of S-phase entry. Prolonged treatments with Br-cAMP or high doses of forskolin (10–100 μm) in the presence of rhGGF2 may lead to the inhibition of components of the MAPK cascade such as Raf, MEK, or MAPK. Other intracellular intermediates such as mTOR or p70 S6K may also be inhibited by long-term treatments. These inhibitory cascades might involve the cAMP-dependent kinase PKA, which can in turn translocate to the nucleus to phosphorylate CREB. An exception occurred for 72 hr treatments with 1 μm forskolin that potentiated the rhGGF2 effect. This effect could be caused by the activation of the Rap1 kinase, which in turn can activate the MAPK cascade, or by stimulation of receptor recycling. The inhibition of the rhGGF2 effect by both forskolin and Br-cAMP could also be mediated via the increase in expression of a cyclin-dependent kinase inhibitor such as p27 KIP1. Brief treatments with forskolin (15 min) or Br-cAMP (1 hr) could increase the density of RTK, such as ErbB receptors, inserted at the membrane surface, allowing the binding of GF such as GGF2. This effect may be partly mediated by PKA-dependent activation and a nonidentified cAMP-dependent pathway. The inhibitors used are shown in boxes. AC, Adenylyl cyclase; CREB, cAMP-responsive element-binding protein;ErbBs, receptor tyrosine kinases of the ErbB family;GF, growth factor; PI3-K, phosphatidylinositol 3-kinase; p70 S6K, the70 kDa S6 protein kinase; RTK, receptor tyrosine kinase.