β-Adrenergic Receptor Trafficking, Degradation, and Cell Surface Expression Are Altered in Dermal Fibroblasts from Hypertrophic Scars

Abstract

Burn trauma elevates catecholamines for up to 2 years and causes hypertrophic scarring. Propranolol, a nonspecific β1-, β2-adrenergic receptor (AR) inverse agonist, counters the hypermetabolic response to elevated catecholamines and may decrease hypertrophic scarring by an unknown mechanism. We investigated the effect of burn injury on β1-, β2-, and β3-AR expression, trafficking, and degradation in human dermal fibroblasts from hypertrophic scar [HSF], non-scar fibroblasts, and normal fibroblasts. We also investigated the modulation of these events by propranolol. Catecholamine-stimulated cAMP production was lower in HSFs and non-scar fibroblasts than in normal fibroblasts. β1- and β2-AR cell surface expression was lowest in HSFs, but propranolol increased cell surface expression of these receptors. Basal β2-AR ubiquitination was higher in HSFs than non-scar or normal fibroblasts, suggesting accelerated receptor degradation. β-AR degradation was mainly driven by lysosomal-specific polyubiquitination at Lys-63 in normal fibroblasts and HSFs, which was abrogated by propranolol. Propranolol also targeted β-AR to the proteasome in HSFs. Confocal imaging showed a lack of β2-AR-GFP trafficking to lysosomal compartments in catecholamine-stimulated HSFs. These data suggest that burn trauma alters the expression, trafficking, and degradation of β-ARs in dermal fibroblasts, which may then affect fibroblast responses to propranolol.

Figure 1. Beta-adrenergic receptor mRNA expression in human dermal fibroblasts treated with or without isoproterenol (ISO) and propranolol (PPL)

a. NF, NSF, and HSF fibroblasts were plated at the density of 6×10⁵ cells per well and harvested 24 hours later to determine mRNA levels for ß1-, ß2-, and ß3-ARs under basal conditions. Messenger RNA levels were determined using qRT-PCR Cycle threshold (CT) values. CT values were pooled from 3 patients and compared in the 3 cell lines. b. Modulation of ß1-, ß2-, and ß3-AR mRNA expression by ISO and PPL was analyzed in NF, HSF, and NSF fibroblasts. Twenty-four hours after being plated, cells were treated with PPL or ISO for 1 hour or 24 hours. Combined treatment groups were treated with PPL for 1 hour, washed, and then treated with ISO for 1 hour or 24 hours. Appropriate controls were treated with the vehicle at the same time points. Data are presented as fold change compared to NFs at a same passage. *p<0.05 compared to the same cell line treated with vehicle control at 1 hour or 24 hours. Data are means ± SEM of 3 independent experiments. Cells were used between passages 6 and 9. These findings were reproduced in dermal fibroblasts derived from 3 different burn patients.

Figure 2. cAMP generation and PKA activation are reduced in burn patient-derived fibroblasts following stimulation with epinephrine (EPI) and isoproterenol (ISO)

Twenty-four hours after being plated, cells were treated with the indicated combination of EPI (10 μM), ISO (10 μM), and propranolol (PPL, 10 μM). 20 minutes post-treatment, cells were collected and Cyclic AMP levels were measured in a. NFs, b. HSFs, and c. NSFs. *p <0.05, **p<0.01 compared to vehicle-treated fibroblasts. d. PKA activity was analyzed in HSFs, NSFs, and NFs as described in Methods. Data are presented as PKA activity per nanogram of tissue. ^##p<0.01 compared to vehicle-treated NFs, *p<0.05 compared to vehicle-treated NFs, ^$p<0.05 compared to vehicle-treated NSFs, ^@@@p<0.001 compared to vehicle-treated HSFs. For each experiment, all cell lines were used at the same passage, which ranged from passage 6 to 9. Data represent the mean ± SEM of 3 independent experiments.

Figure 3. Propranolol (PPL) treatment alters the localization of ß-ARs in human dermal fibroblasts

Twenty-four hours after being plated, dermal fibroblasts were treated with PPL (10 μM) or vehicle for 24 hours. a. Cell-surface biotinylation and Western blotting were performed as detailed in Methods to assess cell-surface expression of ß1-, ß2-, and ß3-AR. b. Densitometric quantification of ß1-, ß2-, and ß3-AR expression, in the presence or absence of PPL treatment, in the total fraction, cell-surface fraction, and cytosolic fraction. c. Quantification of percent cell-surface expression of ß1-, ß2-, and ß3-ARs in NFs, HSFs, and NSFs, as assessed by flow cytometry. Data represent the mean ± SEM of 3 independent experiments. *p<0.05, **p<0.01, ***p<0.001. Dermal fibroblasts from different patients were used between passages 7 and 9.

Figure 4. Effect of burn trauma on endogenous ß-AR ubiquitination in human dermal fibroblasts treated with or without propranolol (PPL)

Cells were seeded on 10-cm plates in serum media and 24 hours later, transfected with 10 μg/ml HA-ubiquitin DNA in Opti-MEM reduced serum media using Lipofectamine 2000 reagent (Invitrogen, Life Technologies) following the manufacturer’s protocol. Five hours after transfection, Opti-MEM was replaced with full serum media. a. Co-immunoprecipitation/Western blot analysis of ubiquitinated ß2-AR in fibroblasts expressing wild-type HA-ubiquitin. b. Representative blots showing ubiquitination of ß2-AR in the presence or absence of PPL in cells expressing wild-type HA-ubiquitin constructs or mutant HA-ubiquitin constructs in which all Lysine residues (Lys) were mutated to Arginine (Arg) except Lys-63 or Lys-48. All lanes were preincubated with the proteasome inhibitor MG132 as described in Methods. c. Densitometric analysis of ß2-AR ubiquitination. Data are mean ± SEM of 3 independent experiments. p<0.05, ***p<0.001. Dermal fibroblasts from different patients were used between passages 6 and 9.

Figure 5. Propranolol (PPL) modulates ß-AR degradation in human dermal fibroblasts

Twenty-four hours after being plated, NF, HSF, and NSF fibroblasts were pretreated for 30 minutes with PPL or vehicle and then treated for the indicated time with a proteasome inhibitor (MG132) (10 μM) or lysosomal inhibitor (leupeptin) (50 μM). Western blotting was performed to determine the expression of ß1-, ß2-, and ß3-ARs in the presence of a. vehicle or b. PPL (10 μM). c. Quantification of ß1-AR band intensity. d. Quantification of ß2-AR band intensity. e. Quantification of ß3-AR band intensity. Data were normalized to control and were presented as mean ± SEM of at least 3 independent experiments. *p<0.05.

Figure 6. Effect of burn trauma on catecholamine-stimulated trafficking and degradation of β2-AR in human dermal fibroblasts

Dermal fibroblasts were plated on glass coverslips one day before transfection with the 10 μg/ml ß2-AR–GFP construct or control vector using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA). a. Immunofluorescent analysis of NF, HSF, and NSF fibroblasts transfected with ß2-AR–GFP (green) and stimulated for 6 hours with isoproterenol (ISO) or ISO with propranolol (PPL). Lysosomal compartments were visualized with the anti-LAMP2A (red). The bottom two rows show the effect of proteasome (MG132) and lysosome inhibition (Leupeptin-Leup) on ISO-stimulated changes in ß2-AR–GFP trafficking. Arrows point to the areas of colocalization of ß2-AR-GFP to the lysosomes. These experiments have been repeated at least 3 times with 15-20 cells imaged in each preparation. Scale bar, 10 μm. b. Quantification of colocalization (Icorr), presented as the mean ± SEM of 9 separate cells. *p<0.05, **p<0.01.