Abstract
Patients with tuberous sclerosis complex (TSC) develop hamartomas containing biallelic inactivating mutations in either TSC1 or TSC2, resulting in mammalian target of rapamycin (mTOR) activation. Hamartomas overgrow epithelial and mesenchymal cells in TSC skin. The pathogenetic mechanisms for these changes had not been investigated, and the existence or location of cells with biallelic mutations (“two-hit” cells) was unclear. We compared TSC skin hamartomas (angiofibromas and periungual fibromas) with normal-appearing skin of the same patient, and we observed more proliferation and mTOR activation in hamartoma epidermis. Two-hit cells were not detected in the epidermis. Fibroblast-like cells in the dermis, however, exhibited allelic deletion of TSC2, in both touch preparations of fresh tumor samples and cells grown from TSC skin tumors, suggesting that increased epidermal proliferation and mTOR activation were not caused by second-hit mutations in the keratinocytes but by mesenchymal–epithelial interactions. Gene expression arrays, used to identify potential paracrine factors released by mesenchymal cells, revealed more epiregulin mRNA in fibroblast-like angiofibroma and periungual fibroma cells than in fibroblasts from normal-appearing skin of the same patient. Elevation of epiregulin mRNA was confirmed with real-time PCR, and increased amounts of epiregulin protein were demonstrated with immunoprecipitation. Epiregulin stimulated keratinocyte proliferation and phosphorylation of ribosomal protein S6 in vitro. These results suggest that hamartomatous TSC skin tumors are induced by paracrine factors released by two-hit cells in the dermis and that proliferation with mTOR activation of the overlying epidermis is an effect of epiregulin.
Keywords: angiofibromas, paracrine, periungual fibromas, TSC1, TSC2
Hamartomas are benign growths composed of cells normally found in an organ or tissue but in abnormal mixture or organization. Clues to the pathogenesis of hamartomas have come from studies of inherited syndromes in which these lesions are characteristic (1). Patients with tuberous sclerosis complex (TSC), an autosomal dominant tumor syndrome affecting ≈1 in 10,000 individuals, develop hamartomas in multiple organs because of a mutation that inactivates one allele of a tumor suppressor gene, either TSC1 or TSC2 (2). The protein products of these genes, hamartin and tuberin, respectively, form a complex that inhibits the mammalian target of rapamycin (mTOR) pathway, which regulates cell growth (3). TSC hamartomas typically show a “second-hit” somatic mutation that inactivates the wild-type allele. Loss of functional hamartin/tuberin in TSC tumors leads to hyperactivation of mTOR signaling and increased cell number and cell size.
Approximately 90% of TSC patients develop skin hamartomas such as facial angiofibromas (AFs) and periungual fibromas (PFs) (4). These tumors are benign but can be disfiguring or cause bleeding and pain. Current treatments are surgical and have the potential to leave scarring (5).
Like other hamartomas, those in TSC skin contain abnormal numbers of several types of cells. In the dermis, there are increased numbers of large stellate fibroblasts, capillaries, and dermal dendritic cells (6–9). The epidermis is acanthotic (i.e., thickened from increased numbers of keratinocytes in the spinous layer). Acanthosis is pronounced in PFs and variable in AFs (7, 8). The epidermis of treated AFs, several months after argon or CO2 laser surgery, no longer appears acanthotic (10, 11).
We sought to determine whether the overgrowth of epidermal and dermal cells in TSC skin hamartomas was caused by second-hit mutations in the epidermis and dermis or by a “two-hit” cell population that induced proliferation of neighboring cells. Second-hit mutations in more than one cell population have been observed in TSC renal angiomyolipomas. These tumors contain blood vessels, smooth muscle cells, and fat cells; loss of heterozygosity (LOH) at the TSC1 or TSC2 locus in all of these cell lineages has been reported (12, 13). However, it plausible that one cell population could influence neighboring cells, especially in light of the many important mesenchymal–epithelial interactions involved in skin development (14), wound healing (15), and skin tumorigenesis (16). Disruptions in these interactions could contribute to the formation of hamartomas in TSC skin and other organs.
Here, we report that fibroblast-like cells in TSC skin tumors, but not epidermal cells, showed allelic deletion of TSC2. Epiregulin, an epidermal growth factor (EGF)-related growth factor, is overexpressed in fibroblast-like cells grown from TSC skin tumors and may induce epidermal changes.
Results
Keratinocyte Proliferation and mTOR Activation in TSC Skin Tumors.
Tissue sections were stained for nuclear antigen Ki-67 as a marker of cell proliferation and phospho-S6 as a marker of mTOR activation. The number of Ki-67-positive cells in the epidermis of TSC AFs and PFs was clearly larger than that in the same patient's normal-appearing skin (Fig. 1). Cells that were positive for Ki-67 were seen mainly in the basal layer of the epidermis, with few apparent in the dermis.
Fig. 1.
The epidermis of TSC skin tumors shows increased staining for Ki-67 compared with normal-appearing skin. Sections of patient's normal skin (A and D), angiofibromas (B and C), and periungual fibromas (E and F) from two TSC patients were stained with anti-Ki-67 antibody, with normal mouse IgG1 (C and F) as negative controls for A and B, and D and E, respectively. (Scale bars, 35 μm.) Results were similar with three angiofibromas and five periungual fibromas from five patients.
Few cells in the patient's normal-appearing skin were positive for phospho-S6. These cells were restricted almost entirely to a thin layer of cells comprising the granular layer of the epidermis; almost no cells in the dermis were positive for phospho-S6 (Fig. 2A). In contrast, the acanthotic epidermis of TSC skin tumors was strongly positive for phospho-S6 (Fig. 2C). The dermis also contained spindle- and stellate-shaped cells positive for phospho-S6 scattered between collagen bundles, with greater numbers located adjacent to the epidermis. Acanthosis in TSC skin tumors was accompanied by both increased keratinocyte proliferation and mTOR activation.
Fig. 2.
Immunoreactivity for phosphorylated ribosomal protein S6 in the epidermis and dermis of TSC skin tumors but not normal-appearing skin. Sections of normal-appearing skin (A and B) or periungual fibromas (C and D) were incubated with rabbit anti-phospho-S6 (1:200) (A and C) or control rabbit IgG) (B and D) and stained with Vectastain ABC-alkaline phosphatase with Vector red substrate). Patterns of staining were similar in three periungual fibromas and four angiofibromas from four patients.
Two-Hit Cells Are Present in the Dermis, but Not the Epidermis, of TSC Skin Tumors.
The pattern of staining for phospho-S6 in TSC skin tumors appeared consistent with the possibility that keratinocytes and/or scattered fibroblast-like cells in the dermis represented two-hit neoplastic cells. To identify these two-hit cells, samples were analyzed for LOH at the TSC1 and TSC2 loci. LOH was not detected in DNA extracted from microdissected epidermis or dermis of three different AFs (data not shown). Because LOH in two-hit cells could be masked by cellular heterogeneity, we turned to interphase fluorescence in situ hybridization (I-FISH), which allows individual nuclei to be scored for allelic deletion of TSC1 or TSC2, based on the presence of only one signal per nucleus rather than the expected two signals per nucleus for each gene. Keratinocytes derived from three AFs did not show allelic deletion of TSC1 or TSC2 (Table 1); almost all nuclei showed two signals each for TSC1 and TSC2, with 0–4% showing only one signal. (In normal tissues, one signal is often observed in a small percentage of cells because of insufficient hybridization or overlapping signals.) Touch preparations of the dermal side of normal-appearing skin and cultured fibroblasts derived from normal-appearing skin from TSC patients showed similar patterns, with 0–8% showing one signal for TSC1 and 2–12% showing a single signal for TSC2. Among touch preparations or cells cultured from AFs and PFs only 0–8% showed one signal for TSC1, but 11–51% of nuclei had one signal for TSC2. These results are consistent with allelic deletion of TSC2 in a fraction of cells located in the dermis of AFs and PFs, but not in the keratinocytes associated with these tumors, which suggested that the epidermal changes in the tumor originated from effects of tumor mesenchymal cells.
Table 1.
I-FISH analysis of TSC1 and TSC2 genes in TSC tumors
Patient | Tissue | Percentage of single FISH signal |
|||
---|---|---|---|---|---|
Touch preparation cells |
Cultured cells |
||||
TSC1 | TSC2 | TSC1 | TSC2 | ||
Keratinocytes | |||||
1 | NL | ND | ND | 2 | 2 |
AF1 | ND | ND | 2 | 4 | |
AF2 | ND | ND | 4 | 6 | |
AF3 | ND | ND | 0 | 2 | |
2 | NL | ND | ND | 0 | 6 |
AF | ND | ND | 6 | 6 | |
3 | NL | ND | ND | 4 | 4 |
AF | ND | ND | 2 | 4 | |
PF | ND | ND | 0 | 0 | |
Fibroblasts | |||||
2 | NL*† | ND | ND | 4 | 8 |
AF*† | ND | ND | 0 | 20 | |
PF*† | ND | ND | 0 | 18 | |
3 | NL† | ND | ND | 2 | 8 |
AF† | 4 | 51 | 2 | 26 | |
4 | NL*† | ND | ND | 0 | 10 |
AF*† | ND | ND | 6 | 20 | |
PF1 | 2 | 19 | 2 | 28 | |
PF2*† | 4 | 22 | 2 | 18 | |
5 | NL | 6 | 4 | 0 | 8 |
PF | 2 | 19 | 0 | 24 | |
6 | NL | 2 | 6 | 0 | 12 |
PF | 2 | 11 | 0 | 12 | |
7 | NL | ND | ND | 0 | 6 |
AF1 | 2 | 23 | 0 | 28 | |
AF2 | 2 | 32 | 2 | 17 | |
AF3 | 4 | 18 | 0 | 17 | |
8 | NL | 0 | 4 | 2 | 2 |
AF | 2 | 20 | ND | ND | |
9 | NL | 0 | 6 | ND | ND |
PF | 2 | 30 | 0 | 35 | |
10 | NL*† | 8 | 4 | 0 | 12 |
AF*† | 8 | 18 | 0 | 28 | |
PF1*† | 2 | 32 | 0 | 26 | |
PF2 | 0 | 26 | 0 | 14 | |
11 | NL† | 6 | 10 | 0 | 8 |
AF1† | 0 | 22 | 0 | 14 | |
AF2 | 2 | 20 | 2 | 18 | |
12 | NL*† | ND | ND | 0 | 4 |
AF*† | ND | ND | 0 | 18 |
NL, normal-appearing skin; AF, angiofibroma; PF, periungual fibroma, ND, not done.
*Gene expression by Affymetrix array for cells in culture.
†Epiregulin gene expression by real-time PCR for cells in culture.
Epiregulin mRNA Content of Fibroblast-Like Cells Derived from TSC Skin Tumors Is Greater Than That of the Patient's Fibroblasts from Normal-Appearing Skin.
RNA from fibroblast-like cells grown from four AFs, three PFs, and normal fibroblasts from four patients were analyzed by using Affymetrix GeneChips. In total, amounts of 22 probe sets representing 20 genes were increased, and 33 probe sets representing 28 genes were decreased by 3-fold or more (tumor vs. normal) in both AFs and PFs [supporting information (SI) Table 3]. The mRNA with the greatest mean elevation in AFs and PFs was epiregulin, which was 11.8-fold that of the patient's normal fibroblasts in AFs and 22.5-fold in PFs. Another mRNA that was highly overexpressed was MCP-1, which was 9.5-fold the control level in AFs and 3.9-fold in PFs, consistent with our earlier experiments using filter-based arrays (17). Epiregulin, an EGF family member, was investigated further because it appeared relevant to the observed epidermal changes. Amounts of epiregulin mRNA measured in samples from 17 patients using real-time PCR were 3.7- to 690-fold the paired controls in AF cells (n = 13, P = 0.001) and 4.5- to 5,660-fold in PF cells (n = 9, P = 0.008; Table 2). The addition of 100 nM rapamycin for 24 h did not significantly change epiregulin mRNA levels (P = 0.84; SI Table 4).
Table 2.
Gene expression of epiregulin in TSC skin tumor cells compared with normal skin fibroblasts from the same patient
Patient | Epiregulin, pg/18S rRNA, ng |
Tumor/normal |
|||
---|---|---|---|---|---|
AF | PF | NL | AF | PF | |
2 | 1.92 | 44.0 | 0.30 | 6.4 | 147 |
3 | 27.0 | 8.19 | 0.079 | 342 | 104 |
4 | 48.0 | 94.0 | 2.13 | 23 | 44 |
10 | 17.9 | 510 | 0.090 | 198 | 5,660 |
11 | 158 | 0.229 | 690 | ||
12 | 68.9 | 0.120 | 574 | ||
13 | 0.70 | 0.19 | 3.7 | ||
14 | 2.25 | 0.023 | 98 | ||
15 | 74.2 | 0.49 | 151 | ||
16 | 79.4 | 17.7 | 4.5 | ||
17 | 217 | 0.050 | 4,360 | ||
18 | 16.9 | 36.3 | 0.032 | 522 | 1,120 |
19 | 125 | 0.262 | 479 | ||
20 | 8.39 | 0.073 | 114 | ||
21 | 16.5 | 0.215 | 77 | ||
22 | 198 | 9.25 | 21 | ||
23 | 8.27 | 0.080 | 103 |
Epiregulin mRNA levels in normal or tumor cells from indicated patients were quantified by real-time PCR in duplicate. Results were normalized to 18S rRNA, and the ratios of amount in tumor cells to that in normal fibroblasts from the same patient were calculated.
Release of Epiregulin Protein by Fibroblast-Like Cells from TSC Skin Tumors.
After incubation of cells in medium containing [35S]methionine for 24 h, samples of medium were subjected to immunoprecipitation with antibodies against epiregulin, which yielded immunoreactive proteins of ≈5 kDa by using medium from fibroblast-like cells of TSC skin tumors but not from patients' normal-appearing skin (Fig. 3 and SI Fig. 6). The addition of unlabeled recombinant epiregulin protein before immunoprecipitation eliminated this band but not other, apparently nonspecific bands. Incubation of cells with EGF did not measurably alter epiregulin release by TSC tumor cells or normal skin fibroblasts (Fig. 3). Searchlight Protein Array analysis showed that the levels of epiregulin in conditioned medium from PF cells of two patients were 12.8 and 43.6 pg/ml, respectively, whereas the protein in conditioned medium from their normal TSC fibroblasts was undetectable. Changes in levels of other EGF family members (EGF, TGF-α, heparin-binding EGF-like growth factor, and amphiregulin) and keratinocyte growth factor, if seen, were inconsistent (data not shown).
Fig. 3.
Immunoprecipitation of epiregulin from medium of TSC skin tumor cells. PF cells or fibroblasts from normal-appearing skin (NL) from the same TSC patient were grown in 10% FBS/DMEM without or with 100 ng/ml EGF and labeled with [35S]methionine for 24 h. Proteins precipitated from conditioned medium with anti-epiregulin antibody or normal rabbit IgG were separated by SDS/PAGE to identify a band corresponding to epiregulin secreted by TSC skin tumor cells. Added recombinant epiregulin competed with radiolabeled endogenous epiregulin; absence of the band indicates antibody specificity.
Keratinocyte Proliferation and Phosphorylation of Ribosomal Protein S6 Enhanced by Epiregulin.
Recombinant epiregulin added in vitro to normal human keratinocytes stimulated cell proliferation in a concentration-dependent manner (Fig. 4), with a maximum 1.9-fold increase in the rate of BrdU incorporation with 20 ng/ml. Treatment of keratinocytes with epiregulin increased phosphorylation of ribosomal protein S6 within 10 min, which persisted at 1 h and decreased to basal levels after 24 h (Fig. 5 A and C). The epiregulin effect was concentration-dependent and maximal at 100 ng/ml (Fig. 5 B and D). Epiregulin increased phosphorylation of p70S6K similar to ribosomal protein S6 (data not shown). The data indicate that epiregulin enhanced keratinocyte proliferation and mTOR activation.
Fig. 4.
Effect of epiregulin on proliferation of human epidermal keratinocytes. Cells were grown and treated with human recombinant epiregulin as described in Materials and Methods. Cell proliferation was quantified by the amount of BrdU incorporation using chemiluminescence ELISA. Data are means ± SD of triplicate experiments. *, P < 0.01 versus control.
Fig. 5.
Effect of epiregulin on phosphorylation of S6 ribosomal protein in keratinocytes. After incubation for 48 h in keratinocyte-SFM (without added EGF and bovine pituitary extract), cells were incubated without or with 100 ng/ml epiregulin for the indicated times (A and C) or without or with epiregulin at the indicated concentrations for 1 h (B and D) before separation of cell proteins by SDS/PAGE and reaction of Western blots with antibodies against S6 and phosphorylated S6. Shown are representative blots (A and B) and corresponding graphs from triplicate experiments (C and D), in which values are expressed as the means ± SD of the ratios of phospho- to total S6 for treated (at each time or concentration) relative to that of control (time = 0 or concentration = 0). *, P < 0.05; **, P < 0.01 vs. control.
Discussion
Increased epidermal thickness of AFs and PFs was associated with increased proliferation of keratinocytes and mTOR activation. Although mTOR activation is characteristic of two-hit populations in other TSC-related tumors (18–21), we found no evidence of second-hit mutations in the epidermal keratinocytes. Instead, allelic deletion of TSC2 was observed in fibroblast-like cells from the AFs and PFs. These results are consistent with immunohistochemical studies of AFs and PFs that showed loss of hamartin or tuberin expression in fibroblastic interstitial but not epidermal cells (22, 23). Therefore, changes in epidermal cells overlying the tumor appear to the result of the influence of associated mesenchymal cells. Consistent with this view, we found that the release of epiregulin by fibroblast-like cells from TSC skin tumors was significantly greater than that by fibroblasts grown from normal skin of the same patients.
Epiregulin is a member of the EGF family that includes EGF, TGF-α, heparin-binding EGF-like growth factor, amphiregulin, and betacellulin. Epiregulin was first purified from conditioned medium of the NIH 3T3/T7 mouse fibroblast-derived cell line (24). Epiregulin is not produced by normal human fibroblasts, but it is expressed by human fibroblasts immortalized by telomerase reverse transcriptase (25), fibroblastic cells in malignant fibrous histiocytomas (26), and, as we show here, fibroblast-like cells from TSC skin tumors. Epiregulin is an autocrine or paracrine factor that is produced by many human cancers (27, 28).
We propose that epiregulin released by fibroblast-like cells in TSC skin tumors stimulates epidermal cell proliferation and acanthosis. This hypothesis is supported by in vitro studies presented here and others showing that epiregulin is mitogenic for keratinocytes (29–31) and stimulates phosphorylation of EGF receptor in keratinocytes (30) and by the in vivo demonstration that application of recombinant epiregulin to murine excisional wounds increased epidermal proliferation and thickness (32). Epiregulin has also been implicated as a contributing factor to the epidermal changes in psoriasis because it is overexpressed by acanthotic psoriatic epidermis (33). Other paracrine factors may also participate in epidermal acanthosis in TSC skin tumors, but we found that other EGF family members and keratinocyte growth factor were inconsistently, if at all, elevated in tumor-derived cells.
The cause of increased epiregulin expression by fibroblast-like cells derived from TSC skin tumors is unclear. It may not be related to mTOR activation resulting from TSC2 loss because rapamycin treatment did not decrease epiregulin mRNA levels. Furthermore, treatment of normal fibroblasts with TSC2 siRNA dramatically decreased tuberin levels, but epiregulin mRNA levels were only 1.5-fold that in cells treated with control siRNA (S.L., Q.F., and T.N.D., unpublished observations). Recombinant EGF (100 ng/ml) increased the levels of epiregulin mRNA ≈50% in TSC skin tumor cells and normal fibroblasts (S.L. and T.N.D., unpublished observations), consistent with the work of others showing stimulation of epiregulin production through the EGFR pathway (30, 34, 35). This finding appears not to account for elevated levels of basal expression in tumors, however, because treatment of TSC skin tumor cells with AG1478, an EGFR inhibitor, or U0126, a MEK inhibitor, did not change epiregulin mRNA levels (S.L., J.-a.W., and T.N.D., unpublished observations). We note that epiregulin expression is directly regulated by the aryl-hydrocarbon receptor (36), one of the genes identified by using the arrays as overexpressed 2.1-fold in TSC tumor cells.
The disorganized overgrowth of hamartomas is suggestive of abnormal continued expression of factors that stimulate growth early in development. Epiregulin is a plausible candidate for a “hamartic” factor in TSC because it is normally expressed early in development and exists in only a few adult cell lineages (24, 37). Persistent expression of epiregulin by fibroblast-like cells in TSC skin tumors would be consistent with a less than normally differentiated fibroblast phenotype. In this regard, it is of interest that expression of epiregulin in the adult mouse cornea is restricted to a stem cell population in the limbal epithelium (38).
Hamartic factors may be expected to play other roles in tissue morphogenesis. Investigation of the functions of epiregulin in organ formation is at an early stage, but it has been reported to be important in nephron morphogenesis (39). Epiregulin and its receptors or downstream effectors, which appear to be involved in the formation of hamartomas in skin and possibly other organs affected by TSC, could be a therapeutic target for treatment of this disease.
Materials and Methods
Tumor Sample and Cell Culture.
Patients meeting clinical criteria for diagnosis of TSC (40) were enrolled in protocol 00-H-0051 approved by the National Heart, Lung, and Blood Institute/National Institutes of Health (NIH) Institutional Review Board. Samples of AFs, PFs, and normal-appearing skin were obtained and bisected, with one portion used for routine pathology and the other used for frozen sections or cell culture. Fibroblasts from normal-appearing skin or fibroblast-like cells from AFs and PFs were isolated from explants as described in ref. 41 and then cultured in DMEM supplemented with 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin. Keratinocytes from TSC skin samples were obtained by using standard methods (42). Keratinocytes were cultured in serum-free medium (keratinocyte-SFM; Invitrogen), supplemented with EGF and bovine pituitary extract. For studies of the effects of epiregulin on proliferation and cell signaling, keratinocytes from foreskins of unidentified normal neonates were used (provided by Jonathan Vogel, National Cancer Institute, NIH).
Immunohistochemical Staining.
Sections were incubated with mouse anti-human Ki-67 monoclonal antibody (1 μg/ml, clone MIB-1; DakoCytomation), rabbit anti-phospho-S6 (1:200; Cell Signaling Technology), or source- and subtype-matched control IgG (DakoCytomation). Sections were stained with a Vectastain Elite ABC kit and 3,3′-diaminobenzidine peroxidase substrate kit or Vectastain ABC-alkaline phosphatase with Vector red substrate (Vector Laboratories) and counterstained with Mayer's hematoxylin solution (Sigma–Aldrich).
I-FISH.
Cells obtained from touch preparations or culture of TSC normal-appearing skin and skin tumors were analyzed for allelic deletion of TSC1 and TSC2 by using I-FISH as was described in ref. 43. At least 50 nuclei were scored for each sample.
Gene Expression Array.
Early passage cells (passages 3–5) were grown until 70–80% confluent. Total RNA was extracted by using an RNeasy minicolumn (Qiagen), and gene expression analysis was performed as described in the Affymetrix expression analysis technical manual by using 10 μg of total RNA. Fragmented, biotinylated cRNA was hybridized to human genome U133A GeneChips (Affymetrix). Fluorescent probes were added, and intensities were quantified with a GeneArray scanner. Arrays that passed initial quality control requirements were analyzed with Affymetrix MAS 5.1. A total of 11 arrays from four patients were analyzed. Each PF and AF were first compared with the respective patient's control cells from behind the ear or the back. Analysis of the intensities showed that differences between the back and ear locations were small compared with differences between controls and tumor tissue. Also, a comparison of results using normalization methods based on individual controls vs. grouped controls showed little effect (with 994 of 1,180 possible probe sets in common). Probe sets that were identified as “present or marginal” by MAS 5.1 were used for subsequent analysis. Lists of probe sets were constructed for evaluation including those with threefold change and/or statistically significant with a false discovery rate <1% by using a Benjamini–Hochberg multiple testing correction.
Real-Time PCR.
Tumor cells or normal fibroblasts grown from AFs, PFs, or normal skin were incubated in 1% FBS/DMEM with or without 100 nM rapamycin for 24 h before isolation of total RNA from the cells by RNeasy mini kit (Qiagen). Epiregulin mRNA levels were quantified by real-time PCR using the TaqMan universal master mix with assay-on-demand epiregulin primers (Applied Biosystems, Hs 00154995_AI), and values were expressed relative to levels in the same cells of 18S rRNA by using assay-on-demand primers Hs 99999901_S1. Real-time PCR was performed by using the GeneAmp 5700 sequence detection system (Applied Biosystems).
Immunoprecipitation and Multiplex Assay.
Epiregulin protein in conditioned medium of TSC skin tumor cells was detected by using metabolic labeling and immunoprecipitation as reported in ref. 30. Briefly, patient normal fibroblasts or skin tumor cells (1 × 106 cells per 10-cm dish) were labeled in 5 ml of methionine-free DMEM containing 10% dialyzed FBS (Invitrogen) and 1 mCi of [35S]methionine (Amersham Pharmacia Biotech). To some, 100 ng/ml EGF was added at the beginning of the labeling. After 24 h, conditioned media were collected, and an aliquot (1.25 ml) of each medium was used for immunoprecipitation with rabbit anti-epiregulin IgG. The immunoprecipitants were subjected to electrophoresis in 18% Tris-glycine gels (Invitrogen) and analyzed by using the FLA-5100 Bio-Imaging System (Fujifilm Medical System, Inc.). Epiregulin protein released by TSC skin tumor cells was quantified by Thermo Scientific SearchLight multiplex assay (Pierce) according to the manufacturer's procedures.
Cell Proliferation.
Keratinocytes from neonatal human foreskins were added to 96-well Costar Black plates with clear bottoms (1 × 104 cells per well). The next day, wells were washed with keratinocyte-SFM without bovine pituitary extract and human recombinant EGF, and cells were incubated for 24 h followed by incubation for 24 h more in the same medium containing the indicated concentrations of recombinant epiregulin. Cells were labeled with 10 μM BrdU for 3 h, and incorporated BrdU was quantified by using a chemiluminescent ELISA kit (Roche), with chemiluminescence measured with a Fluoroskan Ascent FL (Thermo Labsystems).
Western Blot Analysis.
Neonatal human foreskin keratinocytes were plated on 60-mm dishes at 6 × 105 cells in keratinocyte-SFM. The next day, medium was changed to keratinocyte-SFM without EGF and bovine pituitary extract and incubated for 24 h. Cells were treated with epiregulin as indicated and lysed in protein extraction buffer [20 mM Tris (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 20 mM NaF, 2.5 mM Na2P4O7, 1 mM β-glycerophosphate, 1 mM benzamidine, 10 mM p-nitrophenyl phosphate, 1 mM phenylmethylsulfonyl fluoride]. Equivalent samples of total proteins were separated in 10% (wt/vol) polyacrylamide gels and transferred to 0.45-μm Invitrolon PVDF membranes (Invitrogen) before immunoblotting with 5 μg of anti-phospho-S6 ribosomal protein (Ser-235/236) or anti-S6 ribosomal protein antibodies (Cell Signaling), horseradish peroxidase-conjugated anti-rabbit antibodies (GE Healthcare), and a SuperSignal West Pico chemiluminescence detection kit (Pierce). Band intensity was measured by using a Kodak Capture DC 290 imaging system (Eastman Kodak).
Statistical Analysis.
Epiregulin mRNA data were evaluated by using Wilcoxon signed-rank test. Proliferation (chemiluminescence values) and S6 phosphorylation (ratio of phospho- to total S6 on a log scale) were assessed by using two-way ANOVA with the primary variable epiregulin or time adjusted for differences in baseline level among experiments, followed by Dunnett t test to compare each treatment group with control. Significance was defined as P < 0.05.
Supplementary Material
ACKNOWLEDGMENTS.
We thank the Tuberous Sclerosis Alliance and The LAM Foundation for patient referral. We also thank Catherine Jozwik for the radiolabeling labeling of the cells. This work was supported by a Clinical Scientist Development Award from the Doris Duke Charitable Foundation and National Institutes of Health (NIH) Grant 1 R01 CA100907 (to T.N.D.). G.P.-R. and J.M. were supported by the Intramural Research Program, National Heart, Lung, and Blood Institute, NIH.
Footnotes
The authors declare no conflict of interest.
Data deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE9715).
This article contains supporting information online at www.pnas.org/cgi/content/full/0712397105/DC1.
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