The profibrotic and senescence phenotype of old lung fibroblasts is reversed or ameliorated by genetic and pharmacological manipulation of Slc7a11 expression

Abstract

Increased senescence and expression of profibrotic genes in old lung fibroblasts contribute to disrepair responses. We reported that primary lung fibroblasts from old mice have lower expression and activity of the cystine transporter Slc7a11/xCT than cells from young mice, resulting in changes in both the intracellular and extracellular redox environments. This study examines the hypothesis that low Slc7a11 expression in old lung fibroblasts promotes senescence and profibrotic gene expression. The levels of mRNA and protein of Slc7a11, senescence markers, and profibrotic genes were measured in primary fibroblasts from the lungs of old (24 mo) and young (3 mo) mice. In addition, the effects of genetic and pharmacological manipulation of Slc7a11 were investigated. We found that decreased expression of Slc7a11 in old cells was associated with elevated markers of senescence (p21, p16, p53, and β-galactosidase) and increased expression of profibrotic genes (Tgfb1, Smad3, Acta2, Fn1, Col1a1, and Col5a1). Silencing of Slc7a11 in young cells replicated the aging phenotype, whereas overexpression of Slc7a11 in old cells decreased expression of senescence and profibrotic genes. Young cells were induced to express the senescence and profibrotic phenotype by sulfasalazine, a Slc7a11 inhibitor, whereas treatment of old cells with sulforaphane, a Slc7a11 inducer, decreased senescence without affecting profibrotic genes. Like aging cells, idiopathic pulmonary fibrosis fibroblasts show decreased Slc7a11 expression and increased profibrotic markers. In short, old lung fibroblasts manifest a profibrotic and senescence phenotype that is modulated by genetic or pharmacological manipulation of Slc7a11.

INTRODUCTION

Like other aging tissues, the aging lung is less capable of mounting successful repair responses and, consequently, is susceptible to disrepair after lung injury in humans and experimental animals (1–4). Several processes have been implicated in age-related susceptibility to lung disrepair including epithelial cell dysfunction, endoplasmic reticulum stress, mitochondrial dysfunction, and stem cell exhaustion (5, 6). Aging is also associated with oxidative stress, but the mechanisms responsible for this occurrence, the cells driving this process, and how these events promote tissue disrepair after injury in the aging lung remain incompletely elucidated (7).

Aging is associated with progressive oxidation of the redox potential (Eh) for the extracellular thiol-disulfide couple cysteine (Cys) and cystine (CySS), known as Eh Cys/CySS (8, 9), and this oxidation has been associated with the elicitation of mechanisms involved in atherosclerosis, diabetes, macular degeneration, and chronic obstructive pulmonary disease (COPD), among other disorders (10–12). In primary lung fibroblasts, an oxidized Eh Cys/CySS environment leads to increased expression of the profibrotic growth factor transforming growth factor β1 (TGF-β1) and its intracellular signal transducer Smad3 (13), as well as increased expression of fibronectin and type I collagen. Thus, an oxidized Eh Cys/CySS environment leads to a profibrotic phenotype in lung fibroblasts. These cells play a major role in repair processes after lung injury, suggesting that oxidation of Eh Cys/CySS may contribute to disrepair in the aging lung.

In other work, we showed that fibroblasts not only respond to Eh Cys/CySS but they also contribute to its regulation. Specifically, we showed that young murine lung fibroblasts cultured in media with reduced or oxidized Eh Cys/CySS are capable of returning their redox state toward normal levels within a few hours (14). This capacity, however, is greatly impaired in old lung fibroblasts harvested from 24-mo-old mice when compared with young lung fibroblasts obtained from 3-mo-old mice (14). Old fibroblasts produced a more oxidized extracellular environment when compared with fibroblasts obtained from young mice, around 50 mV more oxidized. Considering the potential implications of these findings, we set out to investigate the mechanisms responsible for the defect observed in old lung fibroblasts. We started by submitting young and old primary lung fibroblasts to transcriptome analysis using microarrays (14). This led to the identification of Slc7a11 (solute carrier family 7 member 11), a gene that codes for xCT, a sodium-independent cystine (CySS)-glutamate antiporter that is chloride-dependent and part of system Xc⁻ (15, 16). This antiporter imports CySS and exports glutamate. System Xc⁻ includes a specific light chain (Slc7a11) and a heavy chain (4F2) linked by disulfide bridges (17). We have shown that expression of Slc7a11 and activity of system Xc⁻ decreases with aging (14, 18). Importantly, we showed that old fibroblasts overexpressing the Slc7a11 gene mimic young fibroblasts in such a way that they are better able to regulate their redox state. On the other hand, young fibroblasts silenced for Slc7a11 gene expression act more like old cells in that they have a diminished capacity to regulate their redox state (18).

Together, these observations point to lung fibroblasts as important regulators of redox state, especially the Eh Cys/CySS, and that this ability is dependent on the appropriate expression and function of the CySS transporter Slc7a11/xCT. This led us to hypothesize that alterations in the expression of this transporter in aging are responsible for the profibrotic and senescence characteristics observed in old fibroblasts. Furthermore, we postulated that genetic or pharmacological manipulation of Slc7a11 expression would reverse this phenotype. To test this hypothesis, both profibrotic and senescence markers were examined in young and old primary murine lung fibroblasts genetically manipulated to overexpress or silence Slc7a11, or treated with sulforaphane, a compound known to increase Slc7a11 expression (18), or sulfasalazine, a compound known to block Slc7a11 function (19).

METHODS

Experimental Reagents

The pharmacological agents, sulforaphane and sulfasalazine, were purchased from Sigma-Aldrich (St. Louis, MO). Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/L glucose, antibiotic-antimycotic, and trypsin were purchased from Corning (Corning, NY).

Cell Culture and Treatment

Primary lung fibroblasts were isolated from five young (3–4 mo old) and five old (18–24 mo old) C57BL/6 female mice as previously described (14, 20). This work was approved by the University of Louisville and Thomas Jefferson University institutional animal care committees. Fibroblasts were used between passage numbers 7 and 10 and were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/L glucose supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic solution. Cell viability and cell numbers were measured using Trypan blue stain and the Bio-Rad TC20 Automated Cell Counter (Bio-Rad, Hercules, CA).

For pharmacological treatments, fibroblasts were seeded into six-well plates at a density of 1 × 10⁶ cells/well, cells were allowed to recover from trypsinization for 24 h before treatment with either sulforaphane (5 µM) or sulfasalazine (150 µM) for 6–48 h; the doses were chosen based on prior dose-response experiments (18). Afterward, cells were harvested, RNA and protein were extracted, and profibrotic and senescence markers were measured using real-time quantitative PCR (qPCR), cell staining, or Western blot analysis.

Human Primary Lung Fibroblasts

Human primary lung fibroblasts were obtained from six explanted lung specimens that were procured from six patients with idiopathic pulmonary fibrosis (IPF) at the time of lung transplantation. This work was approved by the University of Louisville institutional human research committee. Controls included non-IPF lung fibroblasts isolated from the uninvolved adjacent tissue of six patients with lung cancer submitted to resection; this work was approved by the institutional research committee at the University of Manitoba. All patients enrolled in the study provided written consent and the study was approved by the appropriate Institutional Research and Ethics Committees. All procedures for fibroblast isolation, culture, mRNA isolation, and RT-PCR have been published. Briefly, the human lung fibroblasts were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic mixture. Fibroblasts with passage numbers before 10 were used in the experiments.

Measurement of Extracellular Redox Potential

Cells were plated in complete media and allowed to attach overnight. The media was then replaced with a defined redox potential of 0 mV. This was prepared by adding 0.2 mM l-methionine, 4 mM l-glutamine, 99.75 μM l-cystine, and 0.5 μM l-cysteine to DMEM without l-methionine, l-cystine, and l-glutamine immediately before adding to cells. After 24 h, conditioned media were removed and combined with an equal volume of ice-cold solution composed of 200 mM boric acid, 10% (wt/vol) perchloric acid, and 20 μM γ-glutamyl glutamate (18). Samples were derivatized with iodoacetic acid and dansyl chloride. Concentrations of redox couples cysteine (Cys) and cystine (CySS) and of glutathione (GSH) and glutathione disulfide (GSSG) were evaluated via HPLC analysis (Waters Corporation, Milford, MA), and the Nernst equation was used to calculate redox potentials from these concentrations as previously reported (12–14).

Genetic Manipulation of Slc7a11

Plasmid transfection was used to overexpress Slc7a11 in old fibroblasts, whereas siRNA was used to knock down Slc7a11 expression in young fibroblasts as previously described (18). Briefly, plasmid DNA encoding the murine Slc7a11 gene (Origene, Technologies, Inc., Rockville, MD) or siRNA to Slc7a11 (Dharmacon, Lafayette, CO) was used for the studies. The pCMV6-Entry mammalian expression vector (Origene, Technologies, Inc., Rockville, MD) or nontargeting control siRNA (Dharmacon, Lafayette, CO) were used as plasmid and siRNA controls, respectively. Fibroblasts were plated into six-well tissue culture plates (8 × 10⁵ cells/well) and allowed to recover from trypsinization for 24 h before transfection. Plasmid DNA (2.5 μg) or siRNA (50 nM) and Lipofectamine 3000 reagent mix (Thermo Fisher Scientific, Waltham, MA) were incubated at room temperature for 15 min before being added to the wells. The cells were cultured for an additional 48 or 72 h before RNA or protein isolation, respectively.

Measurement of mRNA

The Quick-RNA MiniPrep kit (Zymo Research, Irvine, CA) was used for the isolation and purification of RNA from fibroblasts. Briefly, fibroblasts were seeded into six-well plates at a density of 1 × 10⁶ cells/well and allowed to recover overnight. Fibroblasts were then treated with either sulforaphane (0.5–50 µM) or sulfasalazine (150 µM) for 6 h. Afterward, cells were harvested and RNA was isolated. The iScript cDNA Synthesis kit (Bio-Rad, Hercules, CA) was used to generate cDNA, and qPCR was used to measure profibrotic and senescence gene expression levels. The following TaqMan Gene Expression Assays were used: mouse collagen I (Mm00801666-g1), α-smooth muscle actin (Mm00725412_s1), 18 s (Mm03928990_g1), Slc7a11 (Mm00442530_m1), p21 (Mm00432448_m1) p16 (Mm00494449_m1), βgal (Mm00515342_m1), p53 (Mm00502614), Collagen V (Mm00489342_m1), transforming growth factor β (Mm01178820_m1); or human collagen I (Hs00164004_m1), α-smooth muscle actin (Hs00426835_g1), Slc7a11 (Hs00921938_m1), and 18 s (Hs03003631_g1) according to the manufacturer’s instructions. In addition, mRNA expression levels for fibronectin Extra Domain A (EDA) were measured using SYBR green reagent (Bio-Rad, Hercules, CA). Quantitative Real-Time PCR was determined using the StepOnePlus Real-Time PCR System (Applied Biosystems, Wilmington, DE) with the following parameters: 50°C for 2 min, 95°C for 20 s, followed by 40 cycles of 95°C for 1 s and 60°C for 20 s. The use of iTaq Universal SYBR Green Supermix (Bio-Rad, Hercules, CA) was used with the following parameters: 95°C for 3 min, then 40 cycles of 95°C for 15 s and 60°C for 1 min. The mouse fibronectin EDA primer sequences were the following: forward: (5′-AAC GGA GAA ACG CAG CC) and reverse (5′-GAA ACT TGC CCC TGT GG). The mouse Smad3 primer sequences were the following: forward: (5′-GCA TGG ACG CAG GTT CTC) and reverse (5′-TCC CCT CCG ATG TAG TAG). Results were analyzed using the StepOne Software v. 2.3 (Applied Biosystems, Wilmington, DE) and the amplification curves were analyzed by the mathematical equation of the second derivative. The amount of mRNA expression was normalized to the housekeeping gene 18 s mRNA. The 2^−ΔΔCT method was used to calculate relative quantification (21). Data are expressed as means ± SD of 4–5 independent replicates.

Western Blot Analysis

Primary lung fibroblasts isolated from four independent young (3–4 mo old) and old (18–24 mo old) C57BL/6 mice were seeded into six-well plates and allowed to recover from trypsinization for 24 h. Cells were then harvested in radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Waltham, MA) using a scraper and homogenized using a Bead Mill 4 cell disruptor (Thermo Fisher Scientific, Waltham, MA) and the 2 mL Bead kit with 1.4 mm ceramic beads (Omni International, Kennesaw, GA). Total protein concentration was determined using Bradford protein dye reagent (Bio-Rad, Hercules, CA) and quantified using a Beckman DU800 spectrophotometer (Beckman Coulter, Brea, CA) at optical density (OD) 595. Samples (20 µg) were loaded onto a 10% SDS polyacrylamide gel and electrophoresed using a mini-gel system (Bio-Rad, Hercules, CA) at 150 V for 1 h. Afterward, protein was transferred onto 0.2 µm nitrocellulose membrane (Bio-Rad, Hercules, CA) at 25 V for 1.5 h using a Transblot SD semidry transfer cell (Bio-Rad, Hercules, CA). Membranes were air-dried for 15 min and blocked for 1 h at room temperature in Odyssey Blocking Buffer (Li-COR Biosciences, Lincoln, NE). Membranes were then incubated overnight at 4°C with primary antibody against GAPDH (Abcam, No. ab8245 or No. ab9485, 1:5,000), β-actin (Abcam, No. ab8224, 1:5,000 dilution), βgal (Cell Signaling, No. 27918, 1:1,000 dilution). Slc7a11 (Abcam, No. 175186, 1:1,000 dilution), TGF-β1 (Abcam No.ab215715, 1:1,000 dilution), α-smooth muscle actin (Abcam, No. ab124964, 1:1,000 dilution), Collagen type I (Cell Signaling, No. 91144, 1:1,000 dilution), FN EDA (Sigma, No. SAB4200784, 1:1,000 dilution), p21 (Abcam, No. ab188224, 1:1,000 dilution), p53 (Abcam, No. ab61241, 1:1,000 dilution), or p16 (Abcam, No. ab211542, 1:1,000 dilution). Antibodies used in this study were validated for consistency in binding to specific target proteins with the use of positive and negative lysates from cells or tissues known to express the target protein of interest, lysates from knockout cell lines as negative controls, and analysis of bands at the expected molecular weight. Membranes were washed 3 × 10 min with a phosphate-buffered saline (PBS) solution containing 0.2% Tween-20 (PBST) and then incubated with the appropriate secondary antibody (LI-COR, goat anti-rabbit, No. 926–32211, No. 926–68071 or goat anti-mouse, No. 926–68070, No. 926–32210 at 1:5,000 or 1:20,000 dilution) for 1 h at room temperature. Membranes were washed in PBST (3 × 10 min) and then scanned using an LI-COR Odyssey CLx imaging system and analyzed in Image Studio Lite (LI-COR).

Senescence β-Galactosidase Cell Staining

Primary lung fibroblasts isolated from young and old C57BL/6 mice were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/L glucose supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic solution. Cells were seeded onto a 24-well tissue culture plate at a density of 5 × 10⁴ cells/well. Cells were then treated with sulforaphane (5 µM) or sulfasalazine (150 µM) for 24 h. Afterward, media was removed, cells were rinsed with PBS and 1 mL of 1× Fixative Solution (Senescence β-galactosidase Staining kit, 9860, Cell Signaling Technology, Danvers, MA) was added, and cells were fixed at room temperature for 15 min. Cells were then rinsed two times with PBS before 1 mL of the β-galactosidase staining solution was added, plates were sealed with parafilm, and incubated at 37°C overnight. Cells were photographed using an Evos cell imaging microscope system (Thermo Fisher Scientific, Waltham, MA). The total number of nonstained and senescence (SA) β-galactosidase-positive cells was quantified by counting 3–4 random fields/wells using a Nikon bright-field microscope (Melville, NY) at ×20 magnification. Cell counts were averaged from six separate wells from three biological replicates of young primary lung fibroblasts (3–4 mo old) or five biological replicates of old primary lung fibroblasts (18–24 mo old). An average of 2,415 nonstained and senescence (SA) β-galactosidase-positive cells were counted for each treatment. Results were expressed as the average percentage of positive SA β-galactosidase cells (solid arrows) compared with total number of cells.

Analysis of Data

Means plus standard deviations of the mean were calculated for all experimental values. Significance was assessed by using the Student’s t test. All experiments were repeated a minimum of three times with each sample group containing a minimum number of three experimental groups.

RESULTS

Primary Lung Fibroblasts from Old Mice Show a Profibrotic and Senescence Phenotype

We previously showed that the expression of the Slc7a11/xCT transporter is decreased in old lung fibroblasts (14), which prompted additional studies designed to examine the expression of both profibrotic and senescence markers in these cells. Consistent with our previous report, we demonstrate that primary lung fibroblasts harvested from 24-mo-old mice show a significant decrease in the expression of Slc7a11 mRNA and protein when compared with fibroblasts harvested from 3-mo-old young mice (Figs. 1A and 2A). Consequently, these cells maintain a more oxidized extracellular Eh Cys/CySS than cells from young mice (data not shown) (14). The decreased expression of Slc7a11 was associated with increased mRNA expression for TGF-β1 as well as its intracellular signal transducer Smad3 (Fig. 1, B and C). These changes were associated with increased mRNA expression for the myofibroblast transdifferentiation marker α-smooth muscle actin (Fig. 1D), and the extracellular matrix molecules types I and V collagen and fibronectin EDA (Fig. 1, E–G). We then examined for markers of senescence and found that old lung fibroblasts also showed increased mRNA expression of senescence markers p21, p53, and p16 (Fig. 1, H–J). In addition, β-galactosidase mRNA and protein levels were elevated in old lung fibroblasts when compared with young fibroblasts (Figs. 1K and 2I). When evaluated, the protein levels of each of these markers showed the same trends as mentioned for mRNA expression (Fig. 2, A–I). Together, these studies show an inverse correlation between age and expression of Slc7a11, which was associated with increased expression of profibrotic and senescence markers.

Figure 1.

Aging mouse fibroblasts demonstrate a profibrotic phenotype as demonstrated by mRNA expression. A: primary lung fibroblasts harvested from 24-mo-old mice show a significant decrease in the expression of Slc7a11 mRNA when compared with fibroblasts harvested from 3-mo-old young mice. B: old fibroblasts have increased mRNA expression for TGF-β1 as well as its intracellular signal transducer Smad3 (C). D: old fibroblasts also showed increased mRNA expression for the myofibroblast transdifferentiation marker α-smooth muscle actin and the extracellular matrix molecules collagen type I (E), fibronectin EDA (F), and collagen type V (G). Data are expressed as means ± SD of five independent replicates. *P < 0.05 compared with fibroblasts harvested from 3-mo-old young mice. Aging mouse fibroblasts demonstrate a senescence phenotype. Primary lung fibroblasts harvested from 24-mo-old mice show a significant increase in mRNA expression of markers of senescence including p21 (H), and p53 (I), and p16 (J) when compared with fibroblasts harvested from 3-mo-old young mice. In addition, β-galactosidase mRNA (K) levels were also elevated in old lung fibroblasts when compared with young fibroblasts. TGF-β1, transforming growth factor β1.

Figure 2.

Aging mouse fibroblasts demonstrate a profibrotic and senescence phenotype as demonstrated by protein production. Aging mouse fibroblasts show downregulation of Slc7a11 protein (A), which was associated with upregulation of profibrosis markers (B, TGF-β1; C, α-smooth muscle actin; D, collagen I; and E, fibronectin EDA) and senescence proteins (F, p21; G, 53; H, p16, and I, βgal) as determined by Western blotting. β-actin was used for loading control for Slc7a11 protein, whereas Gapdh was used for loading control for all other proteins, n = 4. *P < 0.05 compared with fibroblasts harvested from 3-mo-old young mice. TGF-β1, transforming growth factor β1.

Genetic Manipulation of Slc7a11 Expression Regulates the Profibrotic and Senescence Phenotype

To evaluate the true role of Slc7a11, genetic approaches were used to upregulate or downregulate the cotransporter. In experiments shown in Fig. 3, young fibroblasts were transfected with Slc7a11 siRNA resulting in silencing of the cotransporter (Fig. 3A). As anticipated, downregulation of Slc7a11 resulted in increased mRNA and protein expression of profibrotic and senescence markers (Fig. 3, B–O). In contrast, old fibroblasts were transfected to overexpress Slc7a11. Cells transfected with the expression vector showed increased Slc7a11 expression (Fig. 4, A and J) as well as decreased mRNA and protein expression of profibrotic and senescence markers (Fig. 4, B–I and K–O).

Figure 3.

Silencing of Slc7a11 in young mouse fibroblasts induces the profibrotic and senescence phenotype. A: primary lung fibroblasts harvested from 3-mo-old mice were silenced with siRNA for Slc7a11 and evaluated for mRNA expression of several markers. As expected, the young cells silenced with siRNA for Slc7a11 showed a decrease in Slc7a11 mRNA expression when compared with nontargeting control siRNA-treated fibroblasts. B: silenced young fibroblasts showed increased mRNA expression for TGF-β1. C: silenced young fibroblasts also showed increased mRNA expression for the myofibroblast transdifferentiation marker α-smooth muscle actin and the extracellular matrix molecules collagen type I (D) and fibronectin EDA (E). The mRNA levels were normalized to the 18 s housekeeping gene, n = 4. Data for mRNA expression levels are expressed as means ± SD of five independent replicates. Silencing of Slc7a11 in young fibroblasts induces the senescence phenotype. Young primary lung fibroblasts silenced for Slc7a11 showed a significant increase in mRNA expression of markers of senescence including p21 (F), and p53 (G), and p16 (H) when compared with nontargeting control siRNA-treated fibroblasts. In addition, β-galactosidase mRNA (I) levels were also elevated in young lung fibroblasts silenced for Slc7a11 when compared with control fibroblasts. The mRNA levels were normalized to the 18 s housekeeping gene, n = 4. Evaluation of protein levels via Western blot. Silencing of Slc7a11 in young lung fibroblasts results in downregulation of Slc7a11 protein (J) while upregulating protein levels of α-smooth muscle actin (K) and collagen type I (L) as well as senescence markers p21 (M), p53 (N), and p16 (O). β-actin was used for loading control for Slc7a11 protein, Gapdh was used for loading control for all other protein, n = 4. Young, 3-mo-old fibroblasts transfected with nontargeting control siRNA. Young + siRNA, 3-mo-old fibroblasts transfected with siRNA for Slc7a11. *P < 0.05 compared with fibroblasts transfected with nontargeting control siRNA. TGF-β1, transforming growth factor β1.

Figure 4.

Overexpression of Slc7a11 in aging mouse fibroblasts reverses the profibrotic and senescence phenotype. A: primary lung fibroblasts harvested from 24-mo-old mice were transfected with Slc7a11 overexpression plasmid and evaluated for mRNA expression of several markers. As expected, the cells showed an increase in Slc7a11 mRNA expression when compared with control fibroblasts. B: transfected old fibroblasts showed decreased mRNA expression for TGF-β1. C: transfected old fibroblasts also showed decreased mRNA expression for the myofibroblast transdifferentiation marker α-smooth muscle actin and the extracellular matrix molecules collagen type I (D) and fibronectin EDA (E). Overexpression of Slc7a11 reverses the senescence phenotype. Old primary lung fibroblasts transfected to overexpress Slc7a11 showed a significant decrease in mRNA expression of markers of senescence including p21 (F), and p53 (G), and p16 (H) when compared with control fibroblasts. In addition, β-galactosidase mRNA (I) levels were also reduced in old lung fibroblasts transfected to overexpress Slc7a11 when compared with control fibroblasts. The mRNA levels were normalized to the 18 s housekeeping gene, n = 4. Evaluation of protein levels via Western blot. J: overexpression of Slc7a11 in old fibroblasts shows increased levels of Slc7a11 protein and downregulates protein levels of profibrotic makers α-smooth muscle actin (K) and collagen type I (L) as well as senescence markers p21 (M), p53 (N), and p16 (O) as demonstrated by Western blot. β-actin was used for loading control for Slc7a11 protein, Gapdh was used for loading control for all other protein, n = 4. Data for mRNA expression levels are expressed as means ± SD of five independent replicates. Control, fibroblasts transfected with the empty expression vector. pSlc7a11, fibroblasts transfected with the Slc7a11 overexpression vector. *P < 0.05 compared with control fibroblasts. TGF-β1, transforming growth factor β1.

Pharmacological Manipulation of Slc7a11 Expression Regulates the Profibrotic and Senescence Phenotype

Having examined the relationship between Slc7a11 expression and profibrotic and senescence markers, we set out to investigate if pharmacological manipulation of the Slc7a11 transporter could influence the profibrotic and senescence phenotype, as was observed in the genetic studies. First, we explored if downregulation or blockade of Slc7a11 could increase the expression of profibrotic and senescent markers in young fibroblasts. In these experiments, young lung fibroblasts were exposed to sulfasalazine, a nonsteroidal anti-inflammatory agent previously shown to inhibit Slc7a11 function (19). Sulfasalazine treatment decreased the expression of Slc7a11 mRNA to levels similar to those observed in old fibroblasts (Fig. 5A). As hypothesized, this change was associated with upregulation of the profibrotic markers TGF-β1, Smad3, α-smooth muscle actin, fibronectin EDA, and both type I and type V collagen (Fig. 5, B–G). In addition, treatment of young fibroblasts with sulfasalazine increased the expression of the senescence markers p21, p53, p16, and β-galactosidase resulting in a phenotype similar to that seen in old fibroblasts (Fig. 5, I–K and Fig. 6).

Figure 5.

Young mouse fibroblasts and the effect of sulfasalazine on profibrotic and senescence phenotype. Young mouse lung fibroblasts treated with sulfasalazine (150 μM) for 6 h showed a significant decrease in the expression of Slc7a11 mRNA (A). Treatment of young mouse fibroblasts with sulfasalazine also showed a significant increase in profibrotic markers TGF-β (B), Smad3 (C), α-smooth muscle actin (D), collagen type I (E), fibronectin EDA (F), and collagen type V (G). Data are expressed as means ± SD of five independent replicates. The mRNA levels were normalized to the 18 s housekeeping gene, n = 4. *P < 0.05 compared with nontreated fibroblasts harvested from 3-mo-old young mice. Young mouse fibroblasts and the effect of sulfasalazine on senescence phenotype. Young mouse lung fibroblasts treated with sulfasalazine (150 μM) for 6 h showed increased expression of senescence markers p21 (H), p53 (I), and p16 (J). Young mouse lung fibroblasts exposed to sulfasalazine (150 μM) for 6 h also demonstrate increased β-galactosidase mRNA expression (K). TGF-β, transforming growth factor β.

Figure 6.

Young mouse fibroblasts and the effect of sulfasalazine on senescence-associated β-galactosidase protein. Young mouse lung fibroblasts were exposed to sulfasalazine (150 μM) for 24 h. Afterward, cells were incubated with β-galactosidase staining solution, photographed, and total number of nonstained and senescence β-galactosidase positive cells (solid arrows) was quantified by counting 3–4 random fields/wells using a Nikon brightfield microscope (Melville, NY) at ×20 magnification. Cell counts were averaged from six separate wells from three biological replicates of young primary lung fibroblasts (3–4 mo old). Results are expressed as the average percentage of β-galactosidase positive cells compared with total number of cells. *P < 0.05 compared with nontreated fibroblasts harvested from 3-mo-old young mice.

We then wanted to see if the opposite was true when testing old fibroblasts. For this, we exposed old lung fibroblasts to sulforaphane, an Nrf2 inducer previously shown to increase the expression of Slc7a11 by our group as well as others (18, 22). Sulforaphane exposure increased Slc7a11 mRNA expression in old lung fibroblasts in a dose-dependent manner with maximum stimulation at 5 µM (Fig. 7, A and B). Interestingly, this effect was not associated with a decrease in the profibrotic markers TGF-β1, Smad3, α-smooth muscle actin, fibronectin EDA, and both type I and type V collagen (Fig. 7, C–H). In contrast, sulforaphane exposure decreased the expression of the senescence markers p21, p53, p16, and β-galactosidase (Fig. 7, I–L and Fig. 8). Similar observations were made when testing protein levels under the above conditions with sulforaphane increasing Slc7a11 and decreasing senescence markers, but with no effect in fibrotic proteins (Fig. 9, A–H).

Figure 7.

Aging mouse fibroblasts and the effect of sulforaphane treatment on profibrotic and senescence phenotype. Primary lung fibroblasts harvested from 24-mo-old mice were exposed for 6 h to different doses of sulforaphane, a Nrf2 inducer previously shown to increase the expression of Slc7a11. A: Slc7a11 mRNA expression was significantly increased with 0.5 to 50 μM sulforaphane with 5 μM showing maximum stimulation. Fibroblasts harvested from 24-mo-old mice exposed to 5 μM sulforaphane showed elevated Slc7a11 mRNA expression (B). Exposure to sulforaphane was not associated with a decrease in the profibrotic markers TGF-β (C), Smad3 (D), α-smooth muscle actin (E), collagen type I (F), fibronectin EDA (G), and collagen type V (H). Aging mouse fibroblasts and the effect of sulforaphane treatment on senescence phenotype. Primary lung fibroblasts harvested from 24-mo-old mice were exposed to 5 μM sulforaphane, a Nrf2 inducer previously shown to increase the expression of Slc7a11. The exposure to sulforaphane for 6 h significantly reduced the mRNA expression levels of several markers of senescence including p21 (I), p53 (J), p16 (K), and β-galactosidase mRNA (L). The mRNA levels were normalized to the 18 s housekeeping gene, n = 4. Nrf2, nuclear factor erythroid 2-related factor 2; TGF-β, transforming growth factor β. *P < 0.05 compared with nontreated fibroblasts harvested from 24-mo-old mice.

Figure 8.

Aging mouse fibroblasts and the effect of sulforaphane treatment on senescence-associated β-galactosidase protein. Primary lung fibroblasts harvested from 24-mo-old mice were exposed to sulforaphane (5 μM) for 24 h. Afterward, cells were incubated with β-galactosidase staining solution, photographed, and total number of nonstained and senescence β-galactosidase positive cells (solid arrows) was quantified by counting 3–4 random fields/wells using a Nikon brightfield microscope (Melville, NY) at ×20 magnification. Cell counts were averaged from six separate wells from three biological replicates of young primary lung fibroblasts (3–4 mo old). Results are expressed as the average percentage of β-galactosidase positive cells compared with total number of cells. *P < 0.05 compared with nontreated fibroblasts harvested from 24-mo-old mice.

Figure 9.

Aging mouse fibroblasts and the effect of sulforaphane treatment on profibrotic and senescence protein levels via Western blot. Primary lung fibroblasts harvested from 24-mo-old mice were exposed to sulforaphane (5 μM) for 48 h. Sulforaphane resulted in upregulation of Slc7a11 protein (A), but had little effects on profibrotic makers (B, TGF-β; C, α-SMA; D, collagen type I; E, fibronectin EDA). However, sulforaphane decreased senescence protein markers (F, p21; G, p53; H, p16). β-actin was used for loading control for Slc7a11 protein, whereas Gapdh was used for loading control for all other proteins, n = 4.*P < 0.05 compared with nontreated fibroblasts harvested from 24-mo-old mice. α-SMA, α-smooth muscle actin; TGF-β, transforming growth factor β.

Lung Fibroblasts from Patients with Idiopathic Pulmonary Fibrosis Show Reduced Expression of Slc7a11 and a Profibrotic Phenotype

Because idiopathic pulmonary fibrosis (IPF) is considered a disease of aging, we set out to investigate if IPF lung fibroblasts show alterations in Slc7a11. For this, we studied primary human lung fibroblasts obtained from six explanted lung specimens that were procured from six patients with IPF at the time of transplantation. Controls included non-IPF lung fibroblasts isolated from the uninvolved adjacent tissue of six patients with lung cancer submitted to resection (see Table 1). As predicted, IPF lung fibroblasts showed a significant decrease in Slc7a11 mRNA expression (Fig. 10A) and this was associated with upregulation of collagen type I and α-smooth muscle actin mRNA expression (Fig. 10, B and C, respectively). As previously reported (18), a reduced Slc7a11 expression in fibroblasts was associated with a more oxidized (positive) extracellular redox environment than that in fibroblasts from non-IPF controls when cultured overnight (Fig. 10D). The average extracellular E_h Cys/CySS was −70 mV for non-IPF fibroblasts and −37 mV for IPF fibroblasts. The average extracellular Cys concentration was 4 µM lower for IPF fibroblasts, accounting for the ∼33 mV oxidation of Eh Cys/CySS (Fig. 10E). There was no difference in extracellular CySS concentration between IPF and non-IPF fibroblasts (Fig. 10F). The intracellular E_h GSH/GSSG was not different in IPF versus non-IPF lung fibroblasts (data not shown).

Table 1.

Characteristics of lung fibroblast donors

Patient Number	Age	Sex
IPF-1613	68	Male
IPF-1705	65	Male
IPF-1710	67	Male
IPF-1713	71	Male
IPF-1714	62	Female
IPF-1715	64	Male
Non-IPF-004	69	Female
Non-IPF-005	64	Male
Non-IPF-090	56	Female
Non-IPF-103	80	Female
Non-IPF-128	57	Female
Non-IPF-138	55	Male

IPF, idiopathic pulmonary fibrosis.

Figure 10.

IPF lung fibroblasts show decreased Slc7a11 expression. IPF and non-IPF lung fibroblasts were isolated and cultured for analysis of mRNA levels by RT-PCR (A–C) or determination of extracellular redox potentials (D–F) as described in MATERIALS AND METHODS. A: Slc7a11 mRNA expression was lower in IPF lung fibroblasts. B: Col1A1 mRNA expression was higher in IPF fibroblasts. C: Acta2 mRNA expression was higher in IPF fibroblasts. D–F: Cys, CySS, GSH, and GSSG concentrations were evaluated by HPLC and inserted into the Nernst equation to calculate the Eh Cys/CySS and Eh GSH/GSSG. D: the Eh Cys/CySS was more oxidized in IPF lung fibroblasts. E: extracellular Cys concentrations (µM) were lower in IPF lung fibroblasts. F: there was no difference in extracellular CySS concentration (µM) between IPF and non-IPF fibroblasts. Cys, couple cysteine; CySS, cystine; GSH, glutathione; GSSG, glutathione disulfide; IPF, idiopathic pulmonary fibrosis. *P < 0.05 compared with non-IPF fibroblasts.

DISCUSSION

Lung aging is associated with tissue disrepair after injury, and fibroblasts are major effector cells in this process leading to excessive connective tissue deposition (23, 24). Interestingly, we have shown that fibroblasts are also involved in regulation of the redox state of the Cys/CySS couple and that this process is severely impaired in old fibroblasts (14). This is relevant as Eh Cys/CySS oxidation promotes extracellular matrix expression in vitro (13). Thus, there is a reciprocal relationship between fibroblast profibrotic functions and Eh Cys/CySS, and we believe that defining the mechanisms responsible for this relationship could point to molecules or pathways that when manipulated, may ameliorate or inhibit susceptibility to tissue disrepair after injury in the aging lung.

One potential target involved in regulation of the redox state of the Cys/CySS couple is Slc7a11, a glutamate-CySS antiporter involved in importing CySS into cells where it can be used for the generation of the antioxidant glutathione. Since Slc7a11 expression is decreased in the aging lung, we postulated that this not only impairs redox regulation but also promotes the profibrotic and senescence phenotype manifested in old lung fibroblasts. Consistent with this, we observed an inverse relationship between the expression of Slc7a11 and many of the markers tested. Specifically, decreased Slc7a11 expression in old fibroblasts was associated with increased expression of the profibrotic markers TGF-β1, Smad3, α-smooth muscle actin, fibronectin EDA, and type I and type V collagen and the senescence markers p21, p53, p16, and β-galactosidase. In contrast, young lung fibroblasts showed higher Slc7a11 expression at baseline, which was associated with dampening of profibrotic and senescence marker expression when compared with old lung fibroblasts. Importantly, both profibrotic and senescent markers were downregulated in aging cells after overexpression of Slc7a11, whereas young fibroblasts silenced for Slc7a11 showed upregulation of profibrotic and senescence markers.

Having evaluated the relationship between Slc7a11 expression and profibrotic and senescence phenotypes in young and old primary lung fibroblasts, we set out to determine if these changes can be pharmacologically manipulated. To that end, we exposed cells to two agents, sulforaphane and sulfasalazine. These agents are known to induce the expression or block the function of Slc7a11, respectively. These interventions partly confirmed our prediction. Treatment of young cells with sulfasalazine decreased the expression of Slc7a11 and increased the expression of both senescence and profibrotic markers. Sulforaphane treatment significantly increased Slc7a11 expression in old fibroblasts while concomitantly decreasing the expression of senescence markers. However, sulforaphane did not alter the expression of the profibrotic markers, thereby dissociating senescence mechanisms from those related to fibrosis, perhaps by off-target effects.

Exactly how sulforaphane and sulfasalazine affect Slc7a11 expression is not entirely clear; however, prior studies point to effects on nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor known to regulate the expression of genes involved in detoxification and antioxidant defense (25, 26). Nrf2 binds to the antioxidant response element (ARE) located in the promoter region of certain genes, which in turn activates genes such as the glutathione transferases (GSTs), catalase, heme oxygenase-1 (HO-1), and NAD(P)H quinone oxidoreductase-1 (NQO1) (27–29). Slc7a11 expression is also influenced by the expression of KEAP, an upstream regulator of Nrf2, and by the transcription factors ATF4 and c-Myc (30). Slc7a11 function has been shown to be affected by its phosphorylation by mTORC2 (which inhibits its transport activity) and its association with Slc3A2, which helps stabilize the membrane complex (31). It is noted that sulforaphane has effects on other systems that might have impacted the observations made. For example, sulforaphane has been shown to attenuate bleomycin-induced lung epithelial cell apoptosis as well as inflammatory cell recruitment, which might account for its protective effects in animals (32); how these observations relate to Slc7a11 is unknown. Sulforaphane can also affect proliferation. In keloid fibroblasts, sulforaphane reduced cell growth (33), whereas it inhibited IL-1β-induced proliferation of synovial fibroblasts from patients with rheumatoid arthritis (34). Slc7a11 downregulation results in oxidation of the Eh Cys/CySS and we previously reported that fibroblasts cultured in oxidized Eh Cys/CySS showed increased proliferation (13). Reversal of this process is predicted to decrease proliferation. Sulforaphane has also been shown to delay senescence, which has important implications for our study (35).

One question that remains unanswered is how Slc7a11 expression affects the senescence and profibrotic pathways. We postulate that Slc7a11 acts by influencing Eh Cys/CySS, which, in turn, influences gene expression as we previously demonstrated (13). Thus, interventions targeting Eh Cys/CySS directly might affect downstream intracellular pathways, thereby affecting fibroblast phenotype. Others have demonstrated that the Eh Cys/CySS could be manipulated with diets with distinct sulfur amino acid content in both rodents and humans (36, 37). However, the effects of this intervention on the processes investigated here have not been evaluated. Another possibility is that Slc7a11 expression and/or function affects the development of the senescence-associated secretory phenotype (SASP) with subsequent release of soluble mediators (e.g., IL-6) capable of affecting cellular functions (38). We have also documented that downregulation of Slc7a11 or exposure of fibroblasts to oxidized Eh Cys/CySS results in TGF-β production (this report, 13). TGF-β has been implicated in cellular senescence as TGF-β suppression of adenine nucleotide translocase-2 (ANT2) may induce oxidative stress and DNA damage during the initiation of cellular senescence (39). Integrin β3 is suggested to participate in the same process through activation of TGF-β (40). TGF-β-induced Notch1 can also stimulate cellular senescence (41). Aging-related downregulation of Slc7a11 also results in increased production of several extracellular matrices. Although it is conceivable that some of these molecules (alone or in combination) might promote senescence, their role in this process remains undefined (42). It should be highlighted that the effects observed could be related to redox changes in unique proteins. Consistent with this idea, we recently reported that aging fibroblasts show a distinct redox protein signature affecting proteostasis when compared with young lung fibroblasts. This redox protein signature is greatly influenced by the expression of Slc7a11 [43]. In addition, downregulation of the Slc7a11 cotransporter may decrease CySS import, thereby decreasing glutathione; in turn, glutathione depletion could promote premature senescence as reported for other cells (44).

Finally, we set out to test whether the aging phenotype described above in Results is seen in IPF lung fibroblasts. IPF is a fibrosing lung disorder most frequently seen in patients over age 50 (45). We hypothesized that, similar to aging, downregulation of Slc7a11 expression and Eh Cys/CySS oxidation might occur in human IPF lung fibroblasts, and these changes might promote fibrogenesis. This is exactly what we observed. The changes observed could directly result from aging rather than the disease, but the ages of the IPF and control subjects tested were not different (66.2 ± 3.2 yr vs. 63.5 ± 9.7 yr; P = 0.54). We speculate that this phenotype might render the aging lung susceptible to tissue disrepair after injury. If so, measuring Slc7a11 in cells or tissues, or Eh Cys/CySS in peripheral blood might serve as a biomarker of disease susceptibility (12). Our data also point to potential targets for intervention as we have shown increased Slc7a11 expression with sulforaphane.

A limitation of our studies is that we have not tested these mechanisms in pulmonary cells other than fibroblasts. In fact, there is a paucity of data regarding the role of Slc71a11 and Eh Cys/CySS in lung epithelial cells and resident immune cells. Also, there may be sex-related differences in humans that might impact the mechanisms investigated. In the pharmacological studies, we found that sulforaphane did not entirely reverse the phenotype, but we did not expose the cells to the reagents for longer periods. It is conceivable that downregulation of the profibrotic phenotype, for example, requires more prolonged exposure to sulforaphane. Independent of these limitations, the observations presented here, taken together with our previous work, suggest that fibroblast phenotypic changes that promote tissue disrepair in aging and disease could be manipulated pharmacologically, which if confirmed, opens the possibility for the future testing of these and related agents at the clinic. The usefulness of this approach has already been suggested in studies showing sulforaphane protects fibroblasts from ionizing radiation (29). Moreover, experimental models of disrepair show that bleomycin-induced lung injury is ameliorated by sulforaphane, whereas sulfasalazine induces lung injury (32, 46). Again, it is noted that sulforaphane could affect pathways other than Slc7a11, which could also contribute to the observations made. Finally, in vivo studies evaluating the effects of genetic or pharmacological manipulation of Slc7a11 should be conducted.

In conclusion, the expression of the Slc7a11 transporter in fibroblasts is decreased in aging and in IPF lungs, which not only results in oxidation of the Cys/CySS redox state but also is associated with a profibrotic and senescence phenotype. We speculate that this phenotype promotes tissue disrepair after lung injury. Furthermore, we hypothesize that interventions targeting the expression and/or function of Slc7a11 or its downstream effects via pharmacological, dietary, or related interventions may increase resistance to lung injury and disrepair during aging and disease development.

DATA AVAILABILITY

The data that support this study are available upon request.

GRANTS

This research was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health (NIH) under Grant No. P20GM113226-6176 (to W.H.W.), an NIH P500AA024337 Grant (to W.H.W. and J.R.), and a grant from the University of Louisville School of Medicine (to W.H.W.).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

W.H.W. and J.R. conceived and designed research; J.D.R. and E.T.-G. performed experiments; J.D.R., E.T.-G., Y.Z., W.H.W., and J.R. analyzed data; J.D.R., Y.Z., R.S., W.H.W., and J.R. interpreted results of experiments; J.D.R. and J.R. prepared figures; J.R. drafted manuscript; I.N.Z., V.v.B, D.R.N., B.K., A.J.H., R.S., W.H.W., and J.R. edited and revised manuscript; J.D.R., E.T.-G., Y.Z., I.N.Z., V.v.B, D.R.N., B.K., A.J.H., R.S., W.H.W., and J.R. approved final version of manuscript.