Neutrophil gelatinase–associated lipocalin mediates 13-cis retinoic acid–induced apoptosis of human sebaceous gland cells (original) (raw)
Patient selection. In an effort to understand the early changes in skin histology and gene expression in response to 13-cis RA, patients were recruited to have skin biopsies of their upper backs at baseline and 1 week into treatment. A total of 7 patients that were prescribed isotretinoin by their dermatologist for their severe acne were enrolled in the study after signing informed consent forms. Information regarding patient age, sex, the time of biopsy, and the dose of isotretinoin that patients were receiving at the time of their second biopsy are presented in Table 1.
Isotretinoin patient demographics
Histology suggests decrease in sebaceous gland volume after 1 week of 13-cis RA treatment. H&E staining was performed on sections of patient skin obtained from biopsies of skin from the upper back taken at baseline and at 1 week of treatment. A total of 17 sections at baseline and 19 sections at 1 week taken from 6 patients were analyzed using image analysis software. At baseline, sebaceous glands were characteristically large and multilobular. Although changes in architecture were not obvious at 1 week of isotretinoin treatment, an early decrease in the overall size of the sebaceous glands was suggested. The mean area of sebaceous glands in the baseline samples was 0.23 ± 0.09 mm2 (mean ± SEM) compared with 0.12 ± 0.02 mm2 in the 1 week samples, which was not significant using a paired t test (P = 0.16) (data not shown). Of note is that previous reports noted marked reductions of sebaceous gland size at 16 weeks of treatment (2).
13-cis RA induces apoptosis in patient’s sebaceous glands at 1 week. Our previous work indicated that 13_-cis_ RA induced apoptosis within cultured SEB-1 sebocytes (8). To confirm these findings in vivo, a TUNEL-peroxidase assay was performed on skin biopsies obtained from patients at baseline and at 1 week of 13-cis RA therapy (n = 6 pairs of samples). Patient 7 was omitted from the analysis because no sebaceous glands were captured in the baseline biopsy sections. A 4-fold increase in the percentage of TUNEL-positive cells was noted within the sebaceous gland at 1 week of therapy compared with baseline (45.9% ± 11.9% vs. 13.9% ± 6% TUNEL-positive cells; P = 0.005, paired t test; α = 0.05) (Figure 1). TUNEL staining was strongest in the nuclei of sebocytes in the basal layer of the sebaceous gland and in early differentiated sebocytes adjacent to the basal layer (Figure 1, arrows). Apoptosis was selective for the sebaceous gland, as no apoptosis was detected within the epidermis.
TUNEL staining is increased in patients’ sebaceous glands at 1 week of isotretinoin treatment. Skin sections were obtained from paraffin blocks of 7 patients sampled at baseline and 1 week of isotretinoin and were subjected to TUNEL-peroxidase assay, followed by counterstaining with hematoxylin. At least 2 skin sections from each time point were analyzed from every patient. Patient 7 was omitted from the analysis as no sebaceous glands were found in baseline biopsy sections. Results were quantified by counting TUNEL-positive cells / total cells in sebaceous glands. Representative sections from Patient 4 (Pt. 4) taken at baseline and at 1 week of isotretinoin treatment are shown. TUNEL staining was strongest in the nuclei of sebocytes in the basal layer of the sebaceous gland (arrows) and in early differentiated sebocytes adjacent to the basal layer. Apoptosis was selective for the sebaceous gland, as no apoptosis was detected within the epidermis (not shown). Negative control (NC) consists of sections from a skin specimen treated with DNase I and processed without terminal transferase enzyme. Positive control (PC) consists of sections from a skin specimen treated with DNase I and subjected to the full TUNEL assay. Original magnification, ×400.
Gene expression analysis of skin from patients treated with 13-cis RA revealed significant increases in genes encoding calcium-binding proteins, retinoid signaling molecules, solute carriers, and serine proteases. Gene array expression analysis was performed on patient skin biopsies at baseline and 1 week of isotretinoin therapy in order to gain insight into putative pathways mediating the apoptotic effect of 13-cis RA. Array data was not generated for patient 3 due to insufficient quantity of RNA. Using a false discovery rate (FDR) of 0.05 that corresponds to a 5% chance of false-positive gene changes, 38 genes were significantly upregulated and 5 genes were significantly downregulated by 13-cis RA when compared with baseline (Table 2; see Supplemental Data for a full listing of significantly changed genes; supplemental material available online with this article; doi:10.1172/JCI33869DS1). Many of the significantly upregulated genes are known to be retinoid-responsive genes, including retinoic acid responder 1 (tazarotene induced gene 1 [TIG1_]), cellular retinol-binding protein 1, and cellular retinoic acid–binding protein 2. Additionally, calcium-binding proteins (i.e., S100 proteins), serine proteases, serine protease inhibitors (serpins), LCN2, and solute carriers were significantly affected by 13_-cis RA. Interestingly, LCN2 was among the most highly upregulated genes. It is an IL-17 target gene that has been reported to induce apoptosis in murine lymphocytes (6, 9).
Significantly changed genes in patient skin after 1 week of isotretinoin therapy
Gene expression in SEB-1 sebocytes treated with 13-cis RA is similar to profile the observed in human skin. Gene expression array analysis performed on patient skin biopsies contains a mixed population of cells. To identify sebocyte-specific gene changes induced by 13-cis RA, we performed array analysis in SEB-1 sebocytes on 3 samples treated with 0.1 μM 13_-cis_ RA and 3 samples treated with vehicle. Laser-capture microdissection of sebaceous glands from the biopsy specimens was not practical due to the determination that over 200 sections were required per sample to perform the array analysis without using double amplification protocols. A total of 85 genes (78 different genes) were significantly influenced by 13_-cis_ RA: 58 were upregulated and 27 were downregulated. Select genes whose expression was significantly changed are listed in Table 3. (See Supplemental Data for the full listing of significantly changed genes.) As in the patient samples, LCN2 and the tumor suppressor, TIG1, demonstrated the greatest changes in gene expression. In addition, there were changes in several genes involved in apoptosis and innate immunity such as TNF-α–induced protein 2, TRAIL, interferon regulatory factor 1 (IRF1), interferon-induced proteins, NF_κ_B, Fas death receptor, and TIG3 (also known as retinoic acid inducible gene 1 [_RIG1_]). TIG3 encodes an RNA helicase and represents a key intracellular protein that, like TLR3, can recognize viral double-stranded RNA (dsRNA) (10, 11).
Selected, significantly changed genes in SEB-1 sebocytes after 13-cis RA treatment
Quantitative real-time PCR verification of select genes from array analyses. Sufficient RNA was available from 5 of 7 patients (all but Patients 3 and 4) to verify gene expression changes by quantitative real-time PCR (QPCR) for LCN2, retinoic acid receptor responder 1 (RARRES1, TIG1), psoriasin (S100A7), serine protease inhibitor A3 (SERPINA3), and phospholipase A2 group 7 (platelet activating factor acetyl hydrolase) (Figure 2A). For SEB-1 sebocytes, gene array expression changes were verified by QPCR for LCN2, TIG1, insulin-like growth factor–binding protein 3 (IGFBP3), and GATA transcription factor 3 (GATA3) (Figure 2B). With each of the QPCR analyses, the direction and magnitude of the change in expression for the selected genes were similar to those observed with the gene array analyses.
QPCR verification of gene array changes. In order to verify the direction and magnitude of the changes in gene expression induced by 13-cis RA in the gene microarrays, QPCR was performed using primers to select genes whose expression was significantly changed by 13-cis RA in the array analysis. (A) Comparison of array analysis and QPCR on RNA obtained from patient skin biopsies at baseline and 1 week of 13-cis RA treatment. Data represent the mean ± SEM of the fold change in gene expression as determined by QPCR in 5 subjects compared to array analysis performed in 6 subjects. RARRES1, retinoic acid receptor responder 1; S100A7, psoriasin; SERPINA3, serine proteinase inhibitor A3; PLA2G7, phospholipase A2 group 7. (B) Comparison of array analysis and QPCR on RNA obtained from SEB-1 sebocytes incubated for 72 hours in the presence or absence of 13-cis RA. Data represent the mean ± SEM of the fold change in gene expression as determined by QPCR in 8 samples compared to array analysis performed in 3 samples. QPCR results were analyzed by REST-XL software program and *P < 0.05 was considered significant. IGFBP3, insulin-like growth factor–binding protein 3; GATA3, GATA-binding protein 3; ZBTB16, zinc finger and BTB domain-containing 16.
13-cis RA induces expression of NGAL in patients’ sebaceous glands and in SEB-1 sebocytes. The gene LCN2 encodes the protein known as NGAL. Since LCN2 has been reported to be involved in apoptosis and it was among the most highly upregulated genes in both patient skin and in SEB-1 sebocytes, its protein product, NGAL, was chosen for further study as a putative mediator of the apoptotic action of 13-cis RA. Patient skin biopsies that were taken at baseline and at 1 week of isotretinoin therapy were used to assess NGAL expression and localization using immunohistochemistry (Figure 3). In general, NGAL expression is increased after 1 week of isotretinoin treatment, although the amount of increase varied among the patients (Table 4). Patient 7 was omitted from the analysis because no sebaceous glands were captured in the baseline biopsy sections. Interestingly, in 2 out of the 6 patients (Patients 4 and 6, the only females in the study) a minimal increase in NGAL staining was noted. Of the 6 patients examined, only 1 had NGAL expression in sebaceous glands at baseline (Table 4). In each patient, NGAL expression was detected specifically within the basal layer of the sebaceous gland and upper sebaceous duct in sections from skin taken at 1 week of therapy (Figure 3). NGAL expression was not noted within the epidermis. These results are most intriguing when comparing the pattern of NGAL expression with the pattern of TUNEL-positive cells after isotretinoin treatment. NGAL immunoreactivity and TUNEL-positive cells are both present within the basal layer of the sebaceous gland and are absent from the epidermis.
NGAL expression is increased in sebaceous glands in patients biopsied at 1 week of isotretinoin treatment. Immunohistochemistry using an antibody to NGAL was performed on skin sections taken at baseline and at 1 week of isotretinoin treatment. Sections were incubated overnight with a 1:50 dilution of mouse monoclonal LCN2/NGAL antibody and developed using AEC chromagen (red). All sections were counterstained with hematoxylin. Representative images at baseline and after 1 week isotretinoin from Patients 1 and 2 are shown. An image of the epidermis after isotretinoin treatment from Patient 2 is shown. NGAL was expressed in the sebaceous gland and duct of samples of skin taken at 1 week of isotretinoin therapy. NGAL was not expressed in the epidermis. The amount of NGAL staining varies among patients and individual patient results are shown in Table 4. Negative control consists of normal human skin incubated with normal mouse IgG1 antibody. Original magnification, ×100.
Quantification of NGAL staining for individual patients at baseline and after 1 week isotretinoin therapy
The expression of NGAL within SEB-1 sebocytes was verified by QPCR and western blotting. Expression of LCN2 mRNA was detected after 48 hours of 13-cis RA treatment. 13-cis RA concentrations of 1 nM and greater significantly increased LCN2 mRNA levels to a maximum of approximately 6-fold (with micromolar concentrations) when compared with control (Figure 4A). After 72 hours of treatment with concentrations ranging from 0.1 nM to 1 μM 13-cis RA, NGAL protein expression increased in a dose-dependent manner to a maximum of approximately 10-fold with micromolar concentrations in SEB-1 sebocytes when compared with control (P < 0.05) (Figure 4B).
13-cis RA and ATRA increase LCN2 mRNA and NGAL protein expression in SEB-1 sebocytes. SEB-1 sebocytes were treated with vehicle as a control (C), 13-cis RA (0.1 nM, 1 nM, 10 nM, 0.1 μM, or 1 μM) or ATRA (0.1 nM, 1 nM, 10 nM, 0.1 μM, or 1 μM) for 48 and 72 hours. (A) LCN2 mRNA expression was verified by QPCR after 48 hours of retinoid treatment. Data represent mean ± SEM of the fold change in gene expression as determined by QPCR of 5 independent samples. Statistical analysis was performed with REST-XL software program and considered significant if *P < 0.05. (B) Protein expression was verified by western blot at 72 hours of retinoid treatment. Blots were incubated with primary antibody to NGAL as well as β-actin as a loading control. Blots were analyzed by densitometry and normalized to β-actin. The graph represents normalized fold-change values (mean ± SEM) relative to control for a minimum of 3 independent blots. Statistical analysis was performed with paired t test; *P < 0.05.
The first 1,000 base pairs of the LCN2 promoter were scanned for retinoic acid response elements (RAREs), using the predefined consensus sequences within the Transfac database through the Transcription Element Search System (TESS), and revealed the presence of both RAR- and RXR-binding sites. Prior studies suggest that 13-cis RA acts as a reservoir of all-trans retinoic acid (ATRA) and, that after isomerization to ATRA, acts via binding nuclear retinoic acid receptors (12). Although, in other studies, retinoid receptor-independent modulation of signaling pathways by ATRA, such as binding to PKC, have also been reported (13, 14).
To address this issue, SEB-1 sebocytes were treated with increasing concentrations of ATRA in parallel studies and examined for expression of LCN2 mRNA and NGAL protein. QPCR and immunoblotting show that NGAL expression also increased in a dose-dependent manner with ATRA when compared with control. The level of NGAL expression induced by ATRA is less than that with 13-cis RA (7-fold vs. 10-fold), but the difference between ATRA and 13-cis RA was not significant (Figure 4).
Recombinant NGAL protein induces apoptosis in SEB-1 sebocytes. Additional studies aimed at testing the hypothesis that NGAL mediates apoptosis in sebaceous glands were performed using SEB-1 sebocytes. SEB-1 sebocytes were treated with increasing concentrations of purified recombinant human NGAL (rhNGAL) protein, and a TUNEL assay was performed. After 24 hours of treatment with NGAL, the percentage of TUNEL-positive cells in SEB-1 sebocytes was significantly increased to a maximum of approximately 35% using 1 ng/ml rhNGAL (Figure 5). Interestingly, this level of apoptosis is similar to that achieved with 72 hours of 13-cis RA treatment in previous studies (8).
NGAL increases TUNEL staining in SEB-1 sebocytes. SEB-1 sebocytes were treated with vehicle as a control, 1 pg/ml, 10 pg/ml, 100 pg/ml, 1 ng/ml, or 10 ng/ml of purified rhNGAL protein (R&D Systems) or the same concentrations of human actin protein for 24 hours. (A) Representative images of the TUNEL assay using rhNGAL are shown. Original magnification, ×200. (B) Quantification of the percentage of TUNEL-positive stained cells per treatment at 24 hours. Data represent mean ± SEM; n = 4–8. Parallel experiments to control for nonspecific effects of protein were performed using human actin protein (n = 2). The percentage of TUNEL-positive cells is less than 5% with all concentrations of actin, which is similar to control values. rhNGAL significantly increased TUNEL staining compared to control over a wide range of concentrations with maximal induction noted at 1 ng/ml. Statistical analyses were performed between vehicle control and each treatment concentration of rhNGAL using 2-factor ANOVA with replication; *P < 0.05.
NGAL mediates 13-cis RA–induced apoptosis in human sebocytes. To test the hypothesis that NGAL mediates the apoptotic effect of 13-cis RA on sebocytes, we used siRNA to LCN2 in the presence of 13-cis RA and examined the effect on apoptosis using western blotting for cleaved caspase 3, the activated form of caspase 3.
Initially, using QPCR we were able to demonstrate that the expression of LCN2 mRNA was successfully decreased 15-fold (93%) by the siRNA compared with siCONTROL in SEB-1 cells that were treated for 48 hours with 13-cis RA (Figure 6A). As a control, the specificity of siRNA knockdown was verified using siRNA to GAPDH in parallel samples. GAPDH and LCN2 gene expression was successfully inhibited in their respective samples. In addition, protein levels of NGAL were undetectable by western blotting after 48 and 72 hours of 13-cis RA treatment (Figure 6B).
siRNA to LCN2 specifically knocks down LCN2 expression in SEB-1 sebocytes in the presence of 13-cis RA. SEB-1 sebocytes (2 × 106 cells per 100 μl reaction mixture) were nucleofected with 1 μg siCONTROL, GAPDH (as a specificity control), or LCN2 siRNA. 13-cis RA (0.1 μM) was added 24 hours after nucleofection, and cells were incubated for 48 and 72 hours. Extent of siRNA knockdown of gene expression was verified by QPCR and western blotting for LCN2 and GAPDH after 13-cis RA treatment. GAPDH and LCN2 gene expression were successfully inhibited in their respective samples. (A) QPCR analysis of LCN2 and GAPDH mRNA levels at 48 hours of 0.1 μM 13-cis RA treatment. The expression of LCN2 mRNA was successfully decreased 15-fold by the LCN2 siRNA compared with siCONTROL, whereas expression of GAPDH was minimally affected by siRNA to LCN2. GAPDH mRNA expression was decreased 4-fold by the specific GAPDH siRNA when compared with siCONTROL, whereas siRNA to GAPDH had minimal effects on expression of mRNA for LCN2. Data represent mean ± SEM; n = 6. *P < 0.05 as determined by REST-XL program. (B) Western analysis of GAPDH and NGAL protein levels. NGAL protein expression is decreased to undetectable levels by western blotting at 48 and 72 hours of 0.1 μM 13-cis RA treatment with LCN2 siRNA. Representative blots of 4 independent experiments are shown.
After confirming successful inhibition of LCN2 and NGAL expression by siRNA, the effect of LCN2 on apoptosis was determined. siRNA to LCN2 in the presence of 13-cis RA decreased expression of cleaved caspase 3 to approximately 20% of control levels (corresponding to a 5-fold reduction) (Figure 7). These data indicate that NGAL mediates the apoptotic effect of 13-cis RA in sebocytes.
siRNA to NGAL blocks the induction of cleaved caspase 3 by 13-cis RA in SEB-1 sebocytes. Under siRNA conditions described in Figure 6, immunoblotting revealed that siRNA to LCN2 decreases expression of cleaved caspase 3 by approximately 5-fold compared with siRNA control. Blots were incubated with primary antibodies to cleaved caspase 3 as well as β-actin as a loading control to normalize densitometry values. Three independent scrambled siCONTROL (SCRAM) and LCN2 western blots are shown. p17 and p19 are the cleaved or active fragments of caspase 3. Graph represents the normalized expression of cleaved caspase 3 from 5 independent western blots. These data indicate that NGAL mediates the apoptotic response of SEB-1 sebocytes to 13-cis RA. Statistical analysis was performed with paired t test, mean ± SEM; *P < 0.05.
SEB-1 sebocytes express the receptor for NGAL. Recently, a cell surface receptor for the murine homolog to NGAL, 24p3, was identified in murine pro-B lymphocytic FL5.12 cells. The presence of this receptor (24p3R) is believed to be responsible for cell-specific susceptibility to apoptosis (6). It is possible that NGAL also functions through a cell surface receptor in humans. Based on sequence homology, the human homolog of 24p3R is predicted to be the solute carrier member, SLC22A17 (6). Expression of SLC22A17 mRNA was detected in SEB-1 sebocytes by QPCR analysis (data not shown). Furthermore, 24p3R was detected in SEB-1 sebocytes by immunohistochemistry and western blotting, using an antibody to the mouse 24p3R (Figure 8) (6). Additional studies are required to assess the receptor’s involvement in mediating the actions of NGAL.
SEB-1 sebocytes express the receptor for NGAL. (A) SEB-1 sebocytes were grown under standard conditions and immunocytochemistry was performed using an antibody to the murine 24p3R. Slides were counterstained with hematoxylin. Negative control (NC) was processed with normal rabbit IgG antibody in place of the primary antibody. Scale bar: 50 μm. Immunoreactivity for the 24p3R localizes to the cytoplasm of SEB-1 sebocytes. (B) Western analysis confirms presence of the receptor and indicates 2 receptor isoforms are present in SEB-1 sebocytes: high molecular weight (H.M.W.) and 24p3R long (24p3R-L). Positive ([+]; HEK 293 cell lysate) and negative ([–]; T47D cell lysate) controls provided by are shown. All samples were run on the same gel but were noncontiguous. Blot shown is representative of 3 independent experiments.