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. Author manuscript; available in PMC: 2019 Jul 1.
Published in final edited form as: J Invest Dermatol. 2018 Feb 22;138(7):1645–1655. doi: 10.1016/j.jid.2018.01.037

β-Adrenergic receptor trafficking, degradation, and cell-surface expression are altered in dermal fibroblasts from hypertrophic scars

Amina El Ayadi 1,3, Anesh Prasai 1,3, Ye Wang 1,3, David N Herndon 1,2,3, Celeste C Finnerty 1,2,3
PMCID: PMC6021177  NIHMSID: NIHMS963536  PMID: 29476776

Abstract

Burn trauma elevates catecholamines for up to 2 years and causes hypertrophic scarring. Propranolol, a non-specific ß1, ß2 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 ß1-, ß2-, and ß3-ARs expression, trafficking, and degradation in human dermal fibroblasts from hypertrophic scar [HSF], non-scar fibroblasts [NSFs], and normal fibroblasts [NFs]. We also investigated the modulation of these events by propranolol. Catecholamine-stimulated cAMP production was lower in HSFs and NSFs than in NFs. ß1- and ß2-AR cell-surface expression was lowest in HSF fibroblasts, but propranolol increased cell surface expression of these receptors. Basal ß2-AR ubiquitination was higher in HSF than NSF or NF fibroblasts, suggesting accelerated receptor degradation. ß-AR degradation was mainly driven by lysosomal-specific polyubiquitination at Lys-63 in NF and HSF fibroblasts, which was abrogated by propranolol. Propranolol also targeted ß-AR to the proteasome in HSF fibroblasts. Confocal imaging showed a lack of ß2-AR– GFP trafficking to lysosomal compartments in catecholamine-stimulated HSF fibroblasts. These data suggest that burn trauma alters the expression, trafficking, and degradation of ß-ARs in dermal fibroblasts which may then affect fibroblast responses to propranolol.

Keywords: Beta adrenergic receptors, Burn, trauma, hypertrophic scarring, lysosomal degradation, ubiquitination

INTRODUCTION

Burn trauma is characterized by a massive increase in catecholamines (Jeschke et al., 2011) accompanied by increased inflammation and poor wound healing. These occurrences contribute to the formation of hypertrophic scars (HS) that are painful, itchy, compromise function, and reduce the patient’s quality of life (Finnerty et al., 2016). Propranolol, a non-specific ß1- and ß2-adrenergic receptor (AR) inverse agonist, counters post-burn stress responses driven by the massive increase in systemic catecholamines, including the hypermetabolic response (Finnerty and Herndon, 2013, Herndon et al., 2001). We found that propranolol speeds burn wound healing (Ali et al., 2015), which we hypothesize to be associated with decreased hypertrophic scarring; a preliminary review of patient data shows a reduction in post-burn scarring with propranolol (Finnerty, Herndon, El Ayadi, unpublished data). Nevertheless, the mechanisms underlying scar reduction in propranolol-treated patients are unclear.

ß-ARs are members of the G-protein-coupled receptors (GPCRs). This family of cell-surface proteins facilitates physiological responses to hormones, neurotransmitters, lipids, and peptides. ß-AR signaling depends on the subcellular localization of these receptors and the number present on the cell surface at the time of stimulus. Following activation, GPCRs activate adenylate cyclase to generate cAMP, which subsequently releases the catalytic subunits of protein kinase A (PKA). PKA is responsible for activating many downstream targets and enzymes involved in cell proliferation and migration, two processes that are highly implicated in hypertrophic scarring. GPCR signal transduction is also controlled by agonist-induced desensitization through uncoupling from G-protein activation and by internalization—the process whereby receptors undergo endocytosis and are either recycled to the cell surface or degraded internally (Drake et al., 2006, Hanyaloglu and von Zastrow, 2008).

Following receptor activation, agonist-bound ß2-AR is phosphorylated on the carboxyl tail through activation of the GPCR kinases (GRKs) and engagement of ß-arrestins, leading to short-term ß2-AR desensitization (Sarker et al., 2011). GRK-phosphorylated, ß-arrestin–bound ß2-AR is ubiquitinated on two different domains, the intracellular loop (Lys-63) and the carboxyl tail. This ubiquitination targets ß2-ARs for lysosomal degradation, explaining the global reduction in cellular receptor levels that characterizes long-term desensitization of GPCRs upon prolonged agonist stimulation (Sarker et al., 2011).

Here we determined the effect of burn trauma on ß-AR expression, ubiquitination, trafficking, and degradation. Given our observation that propranolol decreases hypertrophic scarring in burn patients, we also investigated whether propranolol may ameliorate scarring by altering these events. We used primary dermal fibroblasts isolated from hypertrophic scars (HSF) and non-scar biopsies (NSF) from several pediatric burn patients. Normal dermal fibroblasts (NFs) served as a control for burn-induced alterations. To mimic the post-burn increase in catecholamines, we stimulated fibroblasts with the ß-AR agonist epinephrine. We also used the ß-AR agonist isoproterenol, which works similarly as some ß2-AR–specific agonists and induces receptor internalization and desensitization. We assessed mRNA transcript expression, receptor distribution, receptor ubiquitination status with hemagglutinin (HA)-tagged wild type (Lauwers et al., 2009) and mutant ubiquitin constructs (Newton et al., 2008), and receptor trafficking with confocal imaging of GFP-tagged ß2-AR.

RESULTS

To determine whether isolated human skin fibroblasts exhibited established markers of fibroblasts, we conducted flow cytometry and found that NFs, HSFs, and NSFs all expressed the anti-fibroblast marker, CD90, fibronectin, and hyaluronic synthase-2, a critical enzyme for the biosynthesis of the fibroblast marker hyaluronan (Supplemental Fig. 1a). These markers confirmed fibroblasts identity, as opposed to monocyte-derived fibrocytes, monocytes, and macrophages(Pilling et al., 2009).

mRNA expression for ß1-, ß2-, and ß3-ARs was similar in HSFs, NSFs and NFs (Fig. 1a). Primer specificity was confirmed in lung and heart tissue, view their enrichment with ß1- and ß2-ARs (Supplemental Fig. 1b). Given the known effects of propranolol, isoproterenol, or isoproterenol+propranolol on ß-AR activity, we determined the effects of these compounds on ß-AR mRNA expression in these cells. Expression of ß1-AR mRNA transcripts was not affected by propranolol, isoproterenol, or isoproterenol+propranolol in any of the fibroblast lines (Fig. 1b).

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

Figure 1

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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.

ß2-AR mRNA transcripts were decreased in HSFs 1 hour post-treatment with isoproterenol or propranolol+isoproterenol (both p<0.05) and remained decreased at 24 hours with propranolol+isoproterenol (p<0.05). Likewise, ß2-AR mRNA expression in NSFs was decreased 1 hour after isoproterenol treatment (p<0.05) compared to vehicle-treated control. These levels were also decreased 24 hours post-treatment with isoproterenol+propranolol (p<0.05). ß3-AR mRNA expression was decreased in HSFs 24 hours post-treatment with isoproterenol or isoproterenol+propranolol (both p<0.05). Propranolol did not reverse the effects of isoproterenol on ß-ARs mRNA transcripts in any of the cell lines examined.

Catecholamine-induced cAMP generation and PKA activation is dampened in HSF fibroblasts and can be blocked by propranolol

24 hours after plating, dermal fibroblasts were treated with epinephrine or isoproterenol± propranolol for 20 minutes. In all three cell types, epinephrine increased cAMP generation, and this was blocked by propranolol (Fig. 2a, b, and c). Epinephrine-induced cAMP generation was higher in NFs than in burn patient-derived fibroblasts (HSFs and NSFs). Similarly, isoproterenol induced a greater increase in cAMP in NFs than in HSFs and NSFs, and this was blocked by propranolol.

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

Figure 2

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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.

The cAMP-dependent protein kinase, PKA, is activated by the release of the catalytic subunit following an increase in cAMP levels. Basal PKA activity was lower in HSFs and NSFs than NFs (both p<0.01) (Fig. 2d). In NFs, PKA activity was increased by epinephrine (p<0.05) and isoproterenol (p<0.01). These effects were reversed by propranolol. In HSFs, epinephrine+propranolol elevated PKA activity (p<0.001).

Propranolol increases cell-surface expression of ß1- and ß2-AR in HSF fibroblasts

ß1- and ß2-AR cell-surface expression was lower in HSFs than in NFs (Fig. 3a and b). Propranolol significantly increased ß1- and ß2-AR cell-surface expression in HSFs (both p<0.05). Propranolol also increased ß1- and ß2-AR levels in the total fraction (p<0.05) without affecting these levels in the cytosolic fraction (Fig. 3a and b). In all dermal fibroblasts, propranolol failed to alter ß3-AR levels in the cell-surface fraction, the cytosolic fraction, or total homogenates. Flow cytometric analysis revealed decreased ß1-AR expression in NFs 15 minutes after epinephrine treatment (p<0.05) and 3 hours after epinephrine+propranolol treatment (p<0.05). ß2-AR expression in NFs was decreased at 15 minutes post-treatment with epinephrine (p<0.05) (Fig. 3c) and at 15 min and 3 hours post-treatment with propranolol (both p< 0.05). ß3-AR expression in NFs was decreased at 1 hour post-treatment with epinephrine, propranolol (both p<0.001), and epinephrine+propranolol (p<0.05). ß3-AR expression was still lowered 3 hours post-treatment with propranolol (p<0.001) and epinephrine+propranolol (p<0.01). In HSFs, none of the treatments altered the expression of ß1-AR. ß2-AR expression in HSFs increased with propranolol at 1 and 3 hours (both p<0.05) (Fig. 3c) while ß3-AR expression was decreased with propranolol at 1 hour (p<0.05) and with epinephrine at 1 and 3 hours (p<0.05). In NSFs, epinephrine treatment increased ß1-AR at 15 and 1 hours (both p<0.05), and decreased ß2-AR at 15 min (p<0.01). None of the treatments affected ß3-AR expression NSFs (Fig. 3c).

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

Figure 3

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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.

Endogenous β-AR ubiquitination is altered in HSF fibroblasts and can be modulated by propranolol

Ubiquitination regulates ß-AR trafficking and degradation (Xiao and Shenoy, 2011). The nature of the polyubiquitin chain dictates protein targeting to the proteasome, lysosomes, or endocytic system. Basal endogenous ß2-AR ubiquitination was higher in HSFs than NSFs and NFs (Fig. 4a). To determine the nature of polyubiquitin chains regulating ß2-AR trafficking, we used Lys-specific polyubiquitin constructs. As shown in Fig. 4b, basal total ubiquitination (WT-Ub) was higher in HSFs than NFs and NSFs (lane-7 vs. 1 and 13, Fig. 4b and c, both p<0.001). Propranolol increased total ubiquitination in NFs (lane-2 vs. lane 1, p<0.01) but not HSFs (lane-8 vs. 7) or NSFs (lane-14 vs.13) (Fig. 4b and c).

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

Figure 4

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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.

Lys-48 polyubiquitination indicative of proteasome-dependent degradation was decreased by propranolol in NFs (lane-3 vs. 4, p<0.05), while slightly increased in HSFs (lane-9 vs. 10). Lys-63 polyubiquitination indicative of lysosomal-dependent degradation was decreased by propranolol in NFs (lane-5 vs. 6, p<0.05) and HSFs (lane-11 vs. 12, p<0.001) (Fig. 4b, and c), suggesting that propranolol may decrease ß2-AR trafficking to the endocytic pathway.

Propranolol modulates the degradation of ß-AR in human dermal fibroblasts

To analyze the proteasomal- and lysosomal-dependent degradation of ß1-, ß2-, and ß3-AR in burn patients-derived dermal fibroblasts, we treated fibroblasts with the proteasome inhibitor MG132 or the lysosomal inhibitor leupeptin for 3 or 6 hours (Fig. 5a–e). In NFs, HSFs, and NSFs, ß1-AR degradation was reduced by MG132 or leupeptin (all p<0.05), suggesting the involvement of both systems in ß1-AR degradation. With propranolol, ß1-AR degradation through the proteasome was reduced in NFs but not in HSFs or NSFs (both p<0.05) (Fig. 5b and c).

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

Figure 5

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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.

Basal ß2-AR degradation was slower in HSFs and NSFs than in NFs (Fig. 5a and d). Propranolol decreased lysosomal ß2-AR degradation in NFs and NSFs as shown by ß2-AR accumulation in these cells (both p<0.05) (Fig. 5b and d). Propranolol shifted ß3-AR degradation to the lysosomes in NFs, as shown by an increase in ß3-AR protein levels with leupeptin (p<0.05) (Fig. 5b and e). In NSFs, propranolol directed ß3-AR degradation towards the proteasome, as seen by an increase in ß3-AR levels with MG132 (p<0.05) (Fig. 5b and e).

ß2-AR–GFP translocates to lysosomal compartments in NFs and NSF fibroblasts

ß2-AR trafficking was monitored by transfecting fibroblasts with GFP-tagged ß2-AR constructs (Han et al., 2013). ß2-AR was expressed mostly at the cell surface of NFs (Fig. 6a left top panel). Under basal conditions, ß2-AR–GFP co-localization to lysosomal compartments was higher in HSFs than NSFs or NFs (Fig. 6a, top middle panel). Six hours after isoproterenol (10 μM) stimulation of fibroblasts resulted in translocation of ß2-AR to the lysosomal compartments, as shown by the increased correlation index in NFs (p<0.01) and NSFs (p<0.05) but not HSFs (Fig. 6a second row and Fig. 6b). Propranolol decreased these effects, as shown by decreased co-localization to the lysosomes in NFs and NSFs treated with isoproterenol±propranolol (both p<0.05) (Fig. 6a, third row, and Fig. 6b) suggesting trafficking of ß2-ARs to other degradation pathways.

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

Figure 6

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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.

To test such possibility, co-localization studies were repeated in fibroblasts treated with isoproterenol±lysosome or proteasome inhibitors. In NFs, leupeptin resulted in lower lysosomal localization of ß2-AR–GFP than the proteasome inhibitor, confirming the canonical lysosomal-targeting of ß2-AR following isoproterenol stimulation in control cells (Fig. 6a, fourth row, left panel). In NSFs, leupeptin decreased isoproterenol-induced ß2-AR trafficking to lysosomes compared to ISO alone (Fig. 6a, bottom row, right panel), while MG132 increased it (Fig. 6a, fifth row right panel). In HSFs, MG132 did not target ß2-AR to the lysosomes (Fig. 6b, bottom row, middle panel). This result was reproduced using Lysotracker® instead of LAMP-2A (data not shown). ß2-AR trafficking pattern was consistently seen in HSFs from several burn patients, suggesting that ß-2AR is targeted to different degradation machinery following isoproterenol stimulation in these cells and therefore, recycling these receptors to the cell surface takes longer in HSFs than in normal fibroblasts.

DISCUSSION

We have shown that wound healing in burn patients is accelerated by propranolol (Ali et al., 2015). In addition, the severity of hypertrophic scarring, as assessed by the Vancouver scar scale, is significantly lower in propranolol-treated patients than in controls (Finnerty, Herndon, El Ayadi, unpublished data). To understand the mechanism by which propranolol decreases hypertrophic scarring, we analyzed the expression, ubiquitination, trafficking, and degradation of ß-ARs in dermal fibroblasts from burn patients skin and scar and in normal fibroblasts for non-burned controls, all of which were found to express established fibroblast markers (Pilling et al., 2009). Together, our data show that burn trauma alters the expression, trafficking, and degradation of ß1- and ß2-AR but not ß3-AR in dermal fibroblasts and that these changes affect fibroblast responses to propranolol.

Basal expression of ß-AR mRNA transcripts was comparable in HSFs, NSFs, and NFs. Epinephrine- and isoproterenol-induced cAMP generation were lower in burn-derived HSFs and NSFs than NFs. Propranolol significantly decreased epinephrine-and isoproterenol-induced cAMP release from NFs and NSFs and to a lesser extent from HSFs. Basal PKA activity was also lower in burn patient-derived fibroblasts than NFs and was not increased by epinephrine or isoproterenol. These data suggest that expression of ß-ARs at the cell-surface may be involved in the decreased response to stimuli. However, this may not be the sole culprit for decreased cAMP and PKA activity in response to receptor stimulation. While burn patient-derived fibroblasts do express ß-ARs, some GPCR signaling components may not function properly. A similar notion was introduced by Reed and others after demonstration that decreased cAMP in atopic dermatitis may be due to a deficiency in adenylate cyclase (Reed et al., 1976, Wadskov et al., 1979). The levels of cAMP and cAMP effector proteins (e.g. PKA) are decreased in inflammatory diseases such as psoriasis and atopic dermatitis (Brion et al., 1986, Wadskov et al., 1979). Similar alterations may be occurring in dermal fibroblasts derived from burn patients due to the hyper-inflammatory environment. Sustained exposure to catecholamines, mimicking the clinical presentation following severe burn injury, alters cAMP/PKA signaling in ventricular myocytes from rats, leading to dysfunction and hypertrophy (Fields et al., 2016). Decreased cAMP/PKA activity also results from an over-activation of β2AR-coupled Gi proteins therefore preventing the positive inotropic effect of agonist-induced βAR stimulation (Xiao et al., 1999).

We investigated the possibility that HSFs may have fewer ß-ARs at the cell-surface. We found that propranolol significantly increased cell-surface and total ß1- and ß2-AR in HSFs but not NSFs and NFs. This suggests that the receptor is not internalized in HSFs to act on intracellular targets. As a result, cAMP generation and PKA activity are both decreased. Propranolol did not significantly affect expression of cell-surface ß3-AR, which mainly activates non-canonical Gi/Nitric oxide synthase pathways when exposed to high levels of catecholamines(Gauthier et al., 1998).

Decreased cell-surface expression of ß2-AR in HSFs may arise from greater degradation of receptors following internalization rather than reduced recycling to the cell surface. Trafficking of ß2-AR to endocytic compartments, the proteasome system, or lysosomes for degradation is partially controlled by ubiquitination (Drake et al., 2006, Xiao and Shenoy, 2011). Indeed, we found that basal ß2-AR ubiquitination was higher in HSFs than other dermal fibroblast types, suggesting that these receptors are tagged for degradation. We determined which proteolytic system participates in ß-AR degradation using hemagglutinin-tagged ubiquitin mutant constructs that recognize specific polyubiquitin chains (Lys-48 polyubiquitin or Lys-63 polyubiquitin). Polyubiquitin chains conjugated to Lys-29 or Lys-48 of ubiquitin direct substrate proteins to the proteasome for degradation. Polyubiquitination on Lys-63 and monoubiquitination are implicated in receptor endocytosis, trafficking, sorting, translation, and DNA repair (Chiu et al., 2006, Lauwers et al., 2009). Propranolol decreased WT ubiquitination and Lys-63-linked polyubiquitination in HSFs, while increasing Lys-48-linked polyubiquitination. Thus, propranolol may help clear accumulated and possibly aggregated ß2-AR in these cells by targeting them to the proteasome for a quick recycling.

Imaging of GFP-tagged ß2-AR was consistent with protein expression, cAMP, and PKA activity data, suggesting that HSFs have fewer cell-surface receptors. HSFs expressed more ß2-AR in the lysosomal compartments than NFs and NSFs. In the presence of isoproterenol, this expression pattern did not change, consistent with the idea that these cells have fewer receptors at the cell surface and therefore are less responsive to isoproterenol. Propranolol restored ß2-AR expression in NFs and NSFs but not HSFs.

In summary, decreased cell-surface expression and increased ubiquitination of ß2-AR in HSFs suggests that ß2-AR is differently modulated by ubiquitination in these cells than in NFs or NSFs. The post-burn catecholamine surge and subsequent hypertrophic scar development may alter the distribution and trafficking of dermal fibroblast ß-ARs and the response of these receptors to propranolol or other beta blockers. Our data show that propranolol increases cell-surface expression and decreases the general ubiquitination of ß2-AR in an attempt to make more receptors available at the cell surface for drug-mediated signaling. Decreased cell-surface expression leads to decreased cAMP generation, PKA activity, and consequent engagement of downstream signaling pathways. Ongoing studies are investigating the effects of propranolol on signaling pathways involved in propranolol-induced reduction of hypertrophic scarring.

MATERIALS & METHODS

Cell culture

Skin and scar biopsies were collected from patients at 6 or 12 months post-burn, and fibroblast isolation was conducted in serum media as previously described (Zhang et al., 2012). Patients enrolled in the clinical trial were between 0 and 18 years of age at the time of injury, had at least 30% of the total body surface area burned, and required at least one operative intervention. This study was part of a large clinical trial (www.clinicaltrials.gov, NCT00675714) evaluating the outcomes of burn survivors. The study protocol and informed consent forms were in compliance with Declaration of Helsinki Principles and were approved by the Institutional Review Board of the University of Texas Medical Branch (Galveston, TX). In this study, we used skin and scar biopsies from 4 patients aged 6 months to 13.8 years old, with total body surface area burns between 30 and 50%. All patients provided written informed consent. Non-transformed primary neonatal, dermal fibroblasts (NFs) were purchased from ATCC (Manassas, VA, USA). Fibroblasts were grown in DMEM media containing 13% fetal bovine serum, 1% antibiotic/antimycotic containing 10,000 IU/ml penicillin, and 10,000 μg/ml streptomycin. All cells were used between passages 6 and 10. HSFs were compared to the NSFs that were excised from the same patient on the same day. For consistency, all paired fibroblasts (HSFs and NSFs) from the same patient as well as NFs were used at the same passage. Propranolol hydrochloride and isoproterenol were purchased from Tocris (Bristol, UK). Epinephrine was purchased from Sigma (St. Louis, MO, USA).

Western blotting

After treatment, cells were lysed in buffer containing 5 mM EDTA, 750 mM NaCl, 250 mM Tris, 5% Triton X-100, 150 mM phenylmethanesulfonyl fluoride, and 0.1% phosphatase inhibitor. Protein concentration was determined using the bicinchoninic acid method (Thermo- Scientific, Waltham, MA, USA). Fifty micrograms of protein lysate was separated by SDS-PAGE (El Ayadi et al., 2012). Antibody details are provided in Supplemental Table 1. Negative and positive controls were used to characterize the ß1-, ß2-, and ß3-AR specificity in our system. Protein expression was quantified by densitometry (Xiao and Shenoy, 2011) using NIH Image-J software (available at http://imagej.nih.gov/ij/; National Institutes of Health, Bethesda, MA, USA).

Quantitative real-time PCR

Messenger RNA was isolated using the RNeasy Plus mini kit (Qiagen, Valencia, CA, USA) and cDNA produced using a cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). PCR primer sequences are listed in Supplemental Table 2.

Flow cytometry

Intracellular and extracellular staining for fibroblast markers and β-ARs in HSF, NSF, and NF cells were detected using a Becton Dickinson Accuri flow cytometer (BD Biosciences, Franklin Lakes, NJ). Data were analyzed using BD FACSDiva software (BD Biosciences, San Jose, CA, USA). The antibodies used are listed in Supplemental Table 1.

cAMP and PKA activity assays

Intracellular cAMP was measured using a Catchpoint cAMP assay kit (Molecular Devices, Sunnyvale, CA, USA) following the manufacturer’s protocol. The PKA activity was measured in twenty micrograms of protein using the PKA Kinase activity kit (Enzo, cat# ADI-EKS-390A) following the manufacturer’s instructions.

Cell-surface biotinylation

Cell biotinylation assays were conducted as previously described (El Ayadi et al., 2012). Control and propranolol-treated fibroblasts were incubated with 1 mM cell impermeant biotinylation reagent (Sulfo-NHS-SS-Biotin; Pierce) for 3 hours at 4°C then quenched and lysed. 500 micrograms protein were added to a 25-μl bed volume of NeutrAvidin agarose resin (Pierce, Thermofisher, NY, USA) and incubated overnight at 4°C. The cytosolic fraction was then collected before washing the resin with wash buffer containing protease inhibitors. Biotinylated proteins were eluted by boiling NeutrAvidin beads in SDS sample buffer and subjected to Western blot.

Degradation assay

Dermal fibroblasts were treated with MG132 (Calbiochem, Millipore, MA, USA) or Leupeptin (Peptides International, Louisville, KY, USA) (El Ayadi et al., 2012, Sarker et al., 2011). Control cells received DMSO as a vehicle. Cells were collected after 0, 3, and 6 hours. ß-AR protein levels were determined by Western blot.

Ubiquitination assays

ß2-AR ubiquitination was determined by transfecting cells with human influenza hemagglutinin (HA)-tagged ubiquitin plasmid constructs from Addgene (Cambridge, MA, USA): wild type-HA-ubiquitin (Plasmid #17608), Lys 48-HA-Ubiquitin (Plasmid #17605), and Lys 63-HA-Ubiquitin (plasmid # 17606). After 16 hours, cells were treated with MG-132 for 3 hours to induce accumulation of ubiquitinated proteins and harvested in 150 μl lysis buffer containing protease inhibitors (Roche Diagnostics, Germany) and 10 mM deubiquitinating enzyme inhibitor N-ethylmaleimide (Sigma, St. Louis, MO, USA). Five hundred micrograms of protein lysate was pre-cleared before incubation with a ß2-AR antibody for 2 hours at 4°C (El Ayadi et al., 2012). Immune complexes were recovered using 50 μl protein A-sepharose beads (Pierce, Thermofisher, NY, USA). The beads were washed with lysis buffer, boiled with SDS sample buffer, and separated by SDS-PAGE/Western blot.

Immunohistochemistry

ß2-AR constructs were kindly provided by Drs. Sudha Shenoy and Robert Lefkowitz. Twenty-four hours post-transfection, fibroblasts were treated with isoproterenol±propranolol, MG132, or leupeptin. ß2-ARs localize to lysosomes 6 hours after activation with isoproterenol (Drake et al., 2006, Sarker et al., 2011). Thus, 6 hours post-treatment, fibroblasts were fixed with 4% paraformaldehyde in PBS. Lysosomal localization was determined using LysoTracker® (Molecular Probes, Eugene, OR, USA; # L7528) or LAMP2 antibody with an Alexa Fluor 538 rabbit secondary antibody. Receptor trafficking was monitored using a Nikon Ti inverted microscope scope (NIKON, Japan) outfitted for high-resolution multi-color fluorescence microscopy and equipped with a Yokogawa CSU-X1 spinning disk, an Andor camera (iXon3 EMCCD, Andor technology Ltd, Oxford Instruments, UK), and a confocal set-up with 448-nm and 561-nm lasers. All images were taken at the room temperature with a Nikon plan apochromatic 100X oil objective with 1.4NA. Images were acquired using Metamorph software (Molecular Devices, Sunnyvale, CA, USA) and grouped in PowerPoint with no manipulation. ß2-AR–GFP colocalization to lysosomes was determined using the ImageJ Colocalization Colormap plugin.

Statistical analysis

Statistical analysis was conducted using Prism (PRISM 5, Graph Pad software, CA). One way ANOVAs or students t-tests were used as appropriate, to compare controls and treated samples. Significance was accepted at p ≤ 0.05.

Supplementary Material

supplement

Acknowledgments

Sources of Support

The authors thank Dr. Kasie Cole for editing and proofreading of the manuscript. This project was supported by a Shriners Hospitals for Children fellowship to AE (84202) as well as by grants from Shriners Hospitals for Children (71001 to CCF and 80100, 71000, 71008, and 84080 to DNH), the National Institutes of Health (R01 GM112936 to CCF, R01 GM056687, P50 GM060338, and NIDILRR H133A120091 to DNH). The project was also conducted with the support of the UTMB’s Institute for Translational Sciences, supported in part by a Clinical and Translational Science Award (UL1TR000071) from the National Center for Advancing Translational Sciences (NCATS). Studies to continue the elucidation of the effects of ß-AR modulation on post-burn wound healing and scarring are funded via R01 GM112936 from the NIH/NIGMS, “Effects of Chronic Catecholamines Exposure on Post-Burn Scarring” The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. None of the study sponsors had any role in the collection, analysis, or interpretation of data; in the writing of the report; or in the decision to submit the article for publication.

Abbreviations

β-AR

Beta-adrenergic receptors

GPCRs

G-protein-coupled receptors

GRK

G-protein-coupled receptor kinase

HSF

Hypertrophic scar fibroblasts

NSF

Non-scar fibroblasts

NF

Normal fibroblasts

HA

Human influenza hemagglutinin

LAMP-2A

Lysosomal associated protein-2A subunit

Footnotes

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Conflict of Interest

DNH receives royalties from Elsevier. The authors state no other conflicts of interest.

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