A Novel Role of Peripheral Corticotropin-Releasing Hormone (CRH) on Dermal Fibroblasts

Corticotropin-releasing hormone, or factor, (CRH or CRF) exerts important biological effects in multiple peripheral tissues via paracrine/autocrine actions. The aim of our study was to assess the effects of endogenous CRH in the biology of mouse and human skin fibroblasts, the primary cell type involved in wound healing. We show expression of CRH and its receptors in primary fibroblasts, and we demonstrate the functionality of fibroblast CRH receptors by induction of cAMP. Fibroblasts genetically deficient in Crh (Crh−/−) had higher proliferation and migration rates and compromised production of IL-6 and TGF-β1 compared to the wildtype (Crh+/+) cells. Human primary cultures of foreskin fibroblasts exposed to the CRF1 antagonist antalarmin recapitulated the findings in the Crh−/− cells, exhibiting altered proliferative and migratory behavior and suppressed production of IL-6. In conclusion, our findings show an important role of fibroblast-expressed CRH in the proliferation, migration, and cytokine production of these cells, processes associated with the skin response to injury. Our data suggest that the immunomodulatory effects of CRH may include an important, albeit not explored yet, role in epidermal tissue remodeling and regeneration and maintenance of tissue homeostasis

IL-6 and TGF-β1 production from Crh+/+ and Crh−/− fibroblasts.

<p>Primary dermal fibroblasts isolated from <i>Crh+/+</i> and <i>Crh−/−</i> mice were cultured in 24-well plates (10⁵ cells/well). Supernatants were collected and analyzed by ELISA for IL-6 (a) or TGF-β1 (b). * represents statistical difference (P<0.05) between genotypes (n = 3 wells/condition/experiment, at least 3 independent experiments).</p

Enhanced migratory response of Crh−/− fibroblasts.

<p>Fibroblasts isolated from <i>Crh+/+</i> and <i>Crh−/−</i> dermis were allowed to attach on the dish. A clear space was produced in the confluent monolayer, cultured in serum free or serum treated with 10 µg/ml mytomycin and the wounded fibroblast layer was photographed immediately and 24 and 48 h after wounding. The distance of migration from the original borders was determined and is indicated (b) as a percentage of the original distance in a representative experiment. * represents statistical difference (P<0.05) between genotypes. (n = 3 wells/treatment/experiment, at least 4 independent experiments).</p

Proliferative response of Crh+/+ and Crh−/− fibroblasts.

<p>(a) Fibroblasts isolated from <i>Crh+/+</i> and <i>Crh−/−</i> dermis plated at an initial density of 8×10³ cells/well for four days. * represents statistical difference (P<0.05) between genotypes. (n = 4 wells/treatment/experiment, at least 5 independent experiments). (b) Fibroblasts isolated from both genotypes cultured at an initial density of 10⁵ cells/well in serum supplemented or serum free medium for 30 hr and apoptosis was measured by staining cells with Annexin V/Propidium Iodide and flow cytometry. The experiment was performed three times, with representative results of one experiment shown here.</p

Mouse dermal fibroblasts express Crh and its receptors.

<p>(a) Total RNA isolated from whole brain from <i>Crh+/+</i> mice (lane 1), NMF isolated from <i>Crh+/+</i> mice (lane 2) and NMF isolated from <i>Crh−/−</i> mice (lane 3) was subjected to RT-PCR for evaluation of <i>Crh</i> expression. Lane 4 represents a sample without RT-enzyme and lane 5 represents a sample without template. (b) <i>Crf1</i> mRNA expression in NMF isolated from <i>Crh+/+</i> mice (lane 2). Lane 1 represents <i>Crf1</i> mRNA expression isolated from whole brain, while lane 3 represents a sample without RT-enzyme and lane 4 a sample without template. (c) <i>Crf2</i> mRNA expression in NMF isolated from <i>Crh+/+</i> mice (lane 2). Lane 1 represents <i>Crf1</i> mRNA expression isolated from heart, while lane 3 represents a sample without RT-enzyme and lane 4 a sample without template. (d) Effects of antalarmin and astressin-2B on specific [¹²⁵I] Tyr0-sauvagine binding to CRF₁ and CRF₂. Membrane homogenates from <i>Crh+/+</i> and <i>Crh−/−</i> fibroblasts were assayed for specific binding with [¹²⁵I] Tyr0-sauvagine, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021654#s4" target="_blank">Materials and Methods</a>. The bars represent the % decrease of specific binding. The mean ± SEM values are from 3 independent experiments, each performed with duplicate determinations. (e) Effect of CRH on cAMP accumulation in <i>Crh+/+</i> and <i>Crh−/−</i> fibroblasts. Stimulation of cAMP accumulation by CRH was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021654#s4" target="_blank">Materials and Methods</a> in intact cells. The mean ± SEM values are from 3 independent experiments, each performed with duplicate determinations. * represents statistical difference (P<0.05) between genotypes exposed to the same treatment and # represents statistical difference (P<0.05) between different treatments in the same genotype.</p

Effect of the CRH antagonists in human dermal fibroblasts.

<p>(a) HDFs isolated from human foreskin were cultured in 96-well plates (8,000 cells/well) in the presence of 100 nM antalarmin or ethanol (control). Proliferation was measured using either MTT or thymidine incorporation. *: represents statistical difference (P<0.05) between treatments (n = 4 wells/treatment/experiment, at least 3 independent experiments). (b) HDFs were allowed to attach on the 100 mm dish. A clear space was produced in the confluent monolayer, cultured in serum free or serum treated with 10 µg/ml mytomycin and the wounded fibroblast layer was photographed immediately and 24 h after wounding (n = 3 wells/treatment/experiment, at least 3 independent experiments). (c) HDFs were cultured in 24-well plates (10⁵ cells/well) in the presence of 100 nM antalarmin or ethanol (control). Supernatants were collected and analyzed by ELISA. * represents statistical difference (P<0.05) between treatments (n = 3 wells/condition/experiment, at least 3 independent experiments). (d) HDFs were cultured in 96-well plates (8,000 cells/well) in the presence of 1000 nM astressin 2B or ethanol (control). Proliferation was measured with MTT, (n = 4 wells/treatment/experiment, at least 3 independent experiments).</p