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. Author manuscript; available in PMC: 2011 Apr 8.
Published in final edited form as: J Invest Dermatol. 2010 Apr 29;130(8):2031–2040. doi: 10.1038/jid.2010.98

AMPHIREGULIN CARBOXY-TERMINAL DOMAIN IS REQUIRED FOR AUTOCRINE KERATINOCYTE GROWTH

Stefan W Stoll 1,3, Jessica L Johnson 1, Yong Li 1, Laure Rittié 1, James T Elder 1,2
PMCID: PMC3072808  NIHMSID: NIHMS190448  PMID: 20428186

Abstract

The EGF receptor ligand amphiregulin (AREG) has been implicated as an important autocrine growth factor in several epithelial malignancies and in psoriasis, a hyperproliferative skin disorder. To characterize the mechanisms by which AREG regulates autocrine epithelial cell growth, we transduced human keratinocytes (KCs) with lentiviral constructs expressing tetracycline (TET)-inducible small hairpin RNA (shRNA). TET-induced expression of AREG shRNA markedly reduced autocrine ERK phosphorylation, strongly inhibited autocrine KC growth with an efficiency similar to metalloproteinase and EGFR inhibitors and induced several markers of KC differentiation including keratins 1 and 10. Addition of various concentrations of exogenous EGFR ligands to KC cultures reversed the growth inhibition in response to AREG blocking antibodies but not to shRNA-mediated AREG knockdown. Lentivirus-mediated expression of the full-length AREG transmembrane precursor, but not of the AREG extracellular domain, markedly reversed the shRNA-mediated growth inhibition and morphological changes, and strongly reduced the induction of multiple markers of KC differentiation. Taken together, our data demonstrate that autocrine human KC growth is highly dependent on the AREG transmembrane precursor protein and strongly suggest a previously unreported function of the metalloproteinase-processed carboxy-terminal domain of AREG.

Keywords: epidermal growth factor receptor, amphiregulin, keratinocyte, proliferation, differentiation

INTRODUCTION

The epidermal growth factor receptor (EGFR) is the prototypical member of the c-ErbB family of transmembrane receptor tyrosine kinases, which also includes ErbB2, ErbB3 and ErbB4 (Olayioye et al., 2000). Binding of EGFR ligands and neuregulins to ErbB receptors results in receptor dimerization and activation of signaling pathways that regulate a multitude of cellular responses (Iordanov et al., 2002; Kansra et al., 2004). EGFR, ErbB2 and ErbB3 are expressed in normal human keratinocytes (NHK) and skin (De Potter et al., 2001; Marques et al., 1999; Stoll et al., 2001) and EGFR-mediated signaling regulates keratinocyte (KC) migration, proliferation and differentiation (Ferby et al., 2006; Peus et al., 1997; Piepkorn et al., 2003; Pittelkow et al., 1993; Tokumaru et al., 2000).

The mammalian EGFR ligand family is comprised of seven members (Pastore et al., 2008; Sanderson et al., 2006), several of which are expressed in KCs including amphiregulin (AREG) (Cook et al., 1991), epigen (EPGN) (Stoll et al., 2010; Strachan et al., 2001), epiregulin (EREG) (Shirakata et al., 2000), heparin-binding EGF-like growth factor (HB-EGF) (Hashimoto et al., 1994), and transforming growth factor-alpha (TGF-α) (Coffey et al., 1987). EGFR ligands are synthesized as transmembrane precursors with an N-terminal extracellular region that contains the EGF-domain characterized by a motif of six spatially conserved cysteine residues (Sanderson et al., 2006). Mature forms of EGFR ligands are released into the extracellular milieu from their membrane-bound precursors via metalloproteinase (MP)-mediated proteolytic cleavage and various members of the ADAM family including ADAM10 and 17 have been implicated in this process (Hinkle et al., 2004; Peschon et al., 1998; Sahin et al., 2004; Sunnarborg et al., 2002; Yan et al., 2002). However, the exact identities of the MP(s) required for EGFR ligand shedding in KCs are still under investigation.

The expression of at least five EGFR ligands by human KCs raises the question why the KC EGFR system relies on multiple ligands. Findings from multiple laboratories including ours suggest the existence of non-redundant, EGFR-dependent signaling mechanisms that are activated in different cellular contexts. For example, we and others have previously shown that HB-EGF is important for keratinocyte migration (Stoll et al., 2010; Tokumaru et al., 2000). In contrast, autocrine KC proliferation and ERK phosphorylation are selectively inhibited by neutralizing antibodies against AREG (Bhagavathula et al., 2005; Kansra et al., 2004; Stoll et al., 2010).

AREG is synthesized as a 252 amino acid precursor protein, which undergoes post-translational modifications via N-linked glycosylation and proteolytic processing resulting in multiple membrane-bound (16, 26, 28 and 50 kDa) and soluble isoforms (9, 19, 21 and 43 kDa ) (Brown et al., 1998). Its name reflects the finding that it can either stimulate or inhibit the growth of various normal and cancer cell lines (Shoyab et al., 1988). AREG has been implicated in pathologies of various organs including breast, colon, liver and prostate (Katoh and Katoh, 2006). In the skin, AREG, HB-EGF and TGF-α are overexpressed in the hyperproliferative epidermis of psoriatic lesions (Cook et al., 1992; Elder et al., 1989; Stoll and Elder, 1998) and transgenic mice overexpressing AREG develop a psoriasiform dermatosis with many similarities to psoriasis (Cook et al., 2004; Cook et al., 1997). In contrast, mice with transgenic expression of TGF-α or HB-EGF display only limited epidermal hyperplasia with little cutaneous inflammation (Dominey et al., 1993; Vassar and Fuchs, 1991).

The objective of this study was to explore the mechanisms by which AREG promotes autocrine KC proliferation. To this end, we asked whether AREG gene silencing would block KC growth in a fashion that could be reversed by treatment of KCs with exogenous GFs. A more general objective of this study was to establish an experimental system that allows conditional knockdown of the expression of any gene in human KCs.

RESULTS

To address the importance of AREG in KC physiology we established an experimental system that allows lentivirus-mediated conditional knockdown of gene expression in human KCs. Immortalized but non-transformed N-TERT-2G KCs (Dickson et al., 2000) previously infected with a lentiviral construct encoding tetracycline repressor (TR) protein (Stoll et al., 2010) were stably transduced with shRNAs against AREG or EGFP under the control of a tetracycline (TET)-regulated histone H1 promoter, or with a lentivirus construct encoding EGFP under the control of a TET-regulated cytomegalovirus (CMV) promoter. After stable selection with antibiotics, these cell lines were termed N-TERT-TR-shAREG (AREG shRNA), N-TERT-TR-shEGFP (EGFP shRNA), and N-TERT-TR-EGFP (EGFP cDNA).

First, we tested the regulation of the TET-inducible system using N-TERT-TR-EGFP cells. Addition of TET leads to conformational changes of the TR protein, which prevents its binding to the TET operator sequences in the CMV or H1 promoter, thereby resulting in the induction of gene or shRNA expression. As demonstrated by western-blotting and immunofluorescence in Fig. 1A, EGFP expression was strongly repressed by the TET repressor protein in untreated cells, but was rapidly induced upon addition of TET.

Figure 1. AREG expression is strongly inhibited by TET-induced expression of AREG shRNA.

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A. Inducible, lentivirus-mediated gene expression is tightly regulated by TET as assessed by EGFP immunofluorescence and immunoblotting. The photographs in the upper panel were taken with identical exposure times. Scale bar = 200 μm. The “+” and “-“ labels on the immunoblot indicate +/- TET B. AREG mRNA expression. N-TERT-KC were grown under autocrine conditions +/- TET and analyzed by QRT-PCR. Asterisks denote a significant reduction in gene expression relative to untreated controls, p < 0.005, n=2 for shEGFP, n=5 for shAREG.. C and D. AREG protein expression. N-TERT KC were grown under autocrine conditions +/- TET for 48-hours (48 h pre-TX), followed by incubation for an additional 2 to 24 hours in basal KSFM +/- TET. AREG protein levels were analyzed in RIPA extracts (C) or in KC conditioned medium (D) by ELISA as described in Material and Methods. Data are expressed as percent of controls, *= p< 0.02 (C) or p< 0.001 (D) vs untreated controls (n=4 for all conditions, except n=2 for shEGFP at 2 and 14 hours).

We next examined the effect of TET-treatment on AREG gene expression in the N-TERT-TR-shAREG cells by QRT-PCR. As can be seen in Fig. 1B, TET-induced expression of AREG shRNA but not EGFP shRNA for 24 and 48 hours significantly inhibited AREG mRNA levels by more than 83%.

To control for non-specific shRNA effects we also measured the expression of interferon-gamma stress response genes including protein kinase PKR and 2’,5’- oligoadenylate synthetase (2’,5’-OAS) (Hovanessian, 2007) in N-TERT-TR-shAREG cells. TET-induced expression of AREG shRNA did not significantly change transcript levels of these genes (data not shown).

Using AREG-specific ELISAs, we tested the effects of shRNA expression on cell-associated AREG protein levels in N-TERT-TR-shAREG and N-TERT-TR-shEGFP cells. In the absence of TET, both cell lines displayed a similar pattern of AREG protein expression with AREG levels ranging between 3 and 12 ng/mg of total protein lysate at the various times points tested (data not shown). TET-induced expression of AREG shRNA reduced cell-associated AREG protein levels by more than 71% at all time points (p < 0.02). In contrast, no significant reduction of AREG protein levels was observed in response to EGFP shRNA expression (Fig. 1C). Expression of AREG shRNA but not EGFP shRNA also significantly (p < 0.001) reduced shed AREG in KC supernatants by more than 95% at all time points (Fig 1D).

As depicted in Figure 2A, AREG was the most prominent EGFR ligand in both NHK and N-TERT KC with all other EGF family ligands being expressed at significantly lower levels. AREG mRNA expression was found to be highly similar whether TaqMan assays specific for the intracellular domain (AREG #1) or the extracellular domain (AREG #2 and 3) were used. Soluble AREG in the culture medium was also very similar in NHK and N-TERT KCs (Fig. 2B).

Figure 2. EGFR ligand expression and shedding in normal human keratinocytes (NHKs) and N-TERT keratinocytes (KCs).

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KCs were grown to 40% confluence and incubated in basal medium for 24 h. Culture supernatants were collected for ELISA and the cells were lysed for QRT-PCR analysis. A. Relative EGFR ligand transcript levels were analyzed by QRT-PCR using TaqMan gene expression assays (Applied Biosystems) as previously described (Stoll et al., 2010). AREG mRNA expression was analyzed with three different TaqMan assays from Applied Biosystems (assay #1: HS00155832; assay # 2: HS00950668; assay # 3: HS00950669). Data are expressed as percent of 36B4 transcript levels, mean +/- SEM, n= 3, *= p< 0.05 vs all other EGFR ligands. B. KC supernatants conditioned for 24 h were analyzed by ELISA for the presence of shed AREG. Data are expressed as ng of soluble AREG per ml of supernatant.

Using blocking antibodies, we have previously shown that autocrine ERK phosphorylation and proliferation of NHK are mediated by shed AREG (Stoll et al., 2010). To confirm these data, we tested the effects of conditional knockdown of AREG on ERK phosphorylation in KCs incubated in growth factor-free culture medium. As previously described using anti-AREG antibodies in NHK (Kansra et al., 2004), autocrine ERK phosphorylation was markedly inhibited in N-TERT KC by AREG neutralizing antibodies and AREG shRNA, but not by EGFP shRNAs (Fig. 3A). Similar to NHK (Stoll et al., 2010), autocrine ERK phosphorylation in N-TERT-KCs was also strongly reduced in response to the MP-inhibitor GM6001 and the ErbB tyrosine kinase inhibitor (TKI) PD158780 (Fig. 3A). Notably, TET treatment did not alter EGF-induced ERK phosphorylation (Fig. 3B).

Figure 3. Autocrine ERK phosphorylation is reduced in the presence of AREG shRNA.

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N-TERT-TR shAREG or shEGFP were grown under autocrine conditions for 48 h as described in Material and Methods followed by treatments as described below and RIPA lysates were analyzed ERK and phospho ERK western blotting. A. Cells were incubated in fresh M154 medium for 2 to 4 h +/- TET with or without 5 μg/ml AREG neutralizing antibodies or IgG isotype control or 1 μM PD158780, 40 μM GM6001, or 100 ng/ml EGF. B. Cells were incubated in fresh M154 medium +/- TET for 30 min followed by treatment with EGF (20 ng/ml) or AREG (100 ng/ml) for 10 min.

We next examined the effects of AREG knockdown on autocrine KC growth by performing growth assays in the presence or absence of TET with and without EGFR and MP-inhibitors and/or EGFR ligand blocking antibodies. A picture of a typical result is shown in Fig. 4A and quantifications of multiple experiments are shown in Fig. 4B. TET-driven conditional knockdown of AREG reduced KC growth by more than 86% relative to untreated controls. In contrast, TET-treatment of N-TERT-TR-shEGFP (Fig. 4 A, B) or N-TERT-TR cells (data not shown) did not inhibit KC growth. Growth of N-TERT KC was also strongly inhibited by the ErbB TKI PD158780, the anti-EGFR Ab IgG225, and the MP inhibitor GM6001. Surprisingly, and in stark contrast to their effects on autocrine ERK phosphorylation, inhibition of N-TERT growth by shRNA was significantly greater than that by anti-AREG (p < 0.05). Similar to NHK (Stoll et al., 2010), growth of N-TERT-TR-shAREG cells in the absence of TET was not reduced in response to neutralizing antibodies against HB-EGF (Fig. 4A, B) or TGF-alpha (Fig. 4A).

Figure 4. AREG knockdown strongly inhibits autocrine KC growth and promotes KC differentiation.

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Cell growth assays were performed on N-TERT-TR-shAREG or N-TERT-TR-shEGFP +/- TET with and without PD158780 (1 μM), GM6001 (40 μM) or neutralizing antibodies against EGFR or its ligands (each at 5 μg/ml). A. Cells were stained with crystal violet and photographed. Each photograph represents duplicate wells. B. KC growth was evaluated by hemacytometer counting. Data are expressed as cell number, percent of untreated controls, mean +/- SEM, n=2-7, *= p< 0.05, **= p< 0.005 vs untreated controls. C. Morphology of N-TERT-KCs at the end of growth assays. Scale bar = 200 μm. D. QRT-PCR of various markers of KC differentiation. Data are expressed as percent of 36B4 (mean +/- SEM, n=6, *= p< 0.05, **= p< 0.005, ***= p<0.0001 vs (-) TET controls, NS = not significant).

Induction of AREG shRNA but not EGFP shRNA markedly changed KC morphology with the appearance of tightly packed colonies made of many small and refractile cells (Figure 4C). QRT-PCR analysis of RNA from AREG knockdown cells revealed a strong induction (> 100 fold) of several markers of KC differentiation including keratin 1 and 10, involucrin, and loricrin and to a lesser extent transglutaminase 1 (TGM1) (> 8- fold increase, Fig. 4D). In contrast, induction of EGFP shRNA resulted in only minor changes in the expression of these differentiation markers (Figure 4D).

To explore the mechanisms of differential inhibition of KC growth by AREG knockdown versus AREG neutralizing Abs, we asked whether exogenous EGF-like growth factors could rescue the effects of AREG knockdown. To this end, we incubated N-TERT-TR-shAREG KCs in the presence or absence of TET or AREG neutralizing Abs with and without various concentrations of recombinant EGFR ligands. Addition of EGF or AREG to N-TERT-TR-shAREG cells in the absence of TET did not markedly alter autocrine KC growth (Figure S1). Surprisingly, recombinant EGF, HB-EGF, EPGN and/or TGF-α or even 100 ng/ml of AREG could not compensate for the growth inhibition caused by knockdown of endogenously expressed AREG (Fig. 5A). In stark contrast, inhibition of KC growth by anti-AREG Abs could be reversed by EGFR ligands. As can be seen in Fig. 5B, maximum inhibition of N-TERT growth was reached with 5 μg/ml of AREG Abs. Addition of EGF to cultures treated with saturating amounts of anti-AREG antibodies restored cell growth to levels similar to those in untreated controls and prevented the appearance of differentiated cells that were observed in cultures incubated with antibodies alone (Fig. 5C). Notably, EGFR ligand treatment markedly changed KC morphology in AREG knockdown cells, with the appearance of large, flattened and multinucleated cells (Fig. 5C).

Figure 5. Inhibition of autocrine KC growth by AREG shRNA cannot be reversed by addition of recombinant EGFR ligands.

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Cell growth assays were performed using N-TERT-TR-shAREG cells in the presence or absence of EGFR ligands +/- TET or +/- AREG blocking Abs. Cell counts are presented in the presence or absence of TET (A) or AREG blocking Abs (B). Data are expressed as total cell number. Error bars indicate SEM, n=3-8 except EPGN, n=2. All TET-treated conditions in Fig. 5A were significantly reduced (p<0.05) vs (-) TET controls. Asterisks in Panel B indicate significant changes vs controls without anti-AREG or EGF (p<0.05). C. KC cell morphology at the end of the growth assay. Arrows indicate groups of differentiated cells that were observed in cultures incubated with anti-AREG or TET. Scale bar = 200 μm.

The marked differences between the effects of AREG blocking Abs and AREG shRNA prompted us to address the importance of the cell-associated domain of AREG in regulating KC growth and differentiation. To explore this, we stably transduced N-TERT-TR-shAREG cells with a constitutive lentiviral expression vector encoding cDNA for the AREG extracellular domain (ECD) or the full-length AREG transmembrane (TM) precursor. An overview depicting the constructs used, the targeting shRNA, and the approximate location of the TaqMan gene expression assays is provided in Fig. 6A. These constructs were effectively transcribed in the newly generated cell lines (N-TERT-TR-shAREG/ECD and N-TERT-TR-shAREG/TM) in the absence of TET, and were not targeted by the 3’-UTR specific shRNA (+TET) (Fig. 6B). This figure also demonstrates the detection of AREG expression of both constructs with a TaqMan assay specific for the ECD (Hs00950669), whereas assay Hs00155832 only detects the TM precursor.

Figure 6. AREG TM precursor restores KC proliferation and prevents KC differentiation in AREG knockdowns.

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N-TERT-TR-shAREG were infected with lentivirus constructs encoding the AREG ECD or complete AREG (TM) precursor. After stable selection, cells were used in cell growth assays. A. Overview of the protein domain structure of the AREG TM precursor, soluble mature AREG, and AREG expression constructs. The approximate location of the TaqMan gene expression assays from Applied Biosystems are indicated. The shRNA targets a sequence in the 3’ UTR of the AREG mRNA (nucleotide position 998-1019 of Genbank # M30704). B. AREG expression in N-TERT using either expression assays specific for the TM or for both constructs as shown in A (mean +/- SEM, n=4-7, #= p<0.005 vs (-) TET controls, *= p<0.05 and **= p<0.005 vs N-TERT-TR-shAREG with and without TET, respectively). C. Crystal violet staining of growth assays with N-TERT-TR-shEGFP and N-TERT-TR-shAREG cell lines incubated in the presence or absence of TET +/- EGF or AREG. D. KC morphology at the end of the growth assays. Scale bar = 200 μm. E. QRT-PCR analysis of markers of KC differentiation. The data for N-TERT-TR-shAREG are the same as in Fig. 4D and are included here for comparison. Error bars indicate mean +/- SEM, n=4-7, *= p<0.05, **= p<0.005 vs TET-treated N-TERT-TR-shAREG.

Using these constructs, we next examined the effects of AREG topology on autocrine KC growth. Both cell lines grew equally well in the absence of TET (Fig. 6C), however, after TET-induced AREG knockdown, the AREG-TM precursor restored KC growth whereas the AREG ECD was ineffective. As previously shown in Fig. 5B, recombinant EGF (20 ng/ml) or AREG (100 ng/ml) did not improve shAREG-mediated inhibition of KC growth.

Similarly to its effects on N-TERT KC growth, lentivirus-mediated expression of the full-length AREG TM precursor (but not the AREG ECD) rescued normal cell morphology and strongly prevented the formation of small cell colonies with differentiated cells in the presence of TET (Fig. 6D). Consistent with its effects on growth and cell morphology, QRT-PCR analysis indicated that the TM precursor of AREG prevented the TET-induced expression of markers of KC differentiation, including K1 and 10, to a much greater extent than the ECD of AREG alone (Fig. 6E). Given that KC differentiation in culture is associated with reduced expression of EGFR (Boonstra et al., 1985), we also asked whether TET-induced expression of AREG shRNA influences EGFR mRNA levels. As expected, EGFR expression was significantly reduced by AREG shRNA (Fig. S2). Consistent with the partial reversion of the differentiated phenotype by the AREG-TM precursor, EGFR expression remained low even in the presence of the non-targeted AREG constructs (Fig. S2).

DISCUSSION

Autocrine EGFR activation by multiple ligands has been well described in proliferating KCs in vitro (Barnard et al., 1994; Coffey et al., 1987; Hashimoto et al., 1994; Piepkorn et al., 1994; Shirakata et al., 2000), but it has remained unclear why the EGFR signaling network relies on more than one ligand. We have previously shown that distinct EGFR ligands stimulate KC behavior in different cellular contexts, HB-EGF favoring a pro-migratory phenotype of skin KCs as opposed to the autocrine proliferative effect of AREG ligands (Kansra et al., 2004; Stoll et al., 2010). To further delineate the mechanism by which AREG stimulates autocrine KC growth, we established an in vitro system that allows conditional knockdown of gene expression in human KCs. Using a TET-inducible EGFP expression construct, we demonstrate that this system is tightly regulated, and that TET-induced expression of AREG- but not EGFP shRNAs strongly reduces AREG mRNA and protein expression in N-TERT-KCs (Fig. 1). The proteolytic release of soluble EGFR ligands from their membrane-bound precursors is important for their proper function (Sanderson et al., 2006). Our data also demonstrate that TET-induced expression of AREG shRNA strongly reduces the presence of soluble AREG in KC supernatants by more than 90%. The use of 1 μg/ml TET in our experiments had no inhibitory effect on AREG shedding as shown in our control cells (Fig. 1D, N-TERT-TR-shEGFP). Together, the results in Fig. 1 strongly validate the TET-inducible lentivirus system as a powerful tool to study gene function in human KCs.

In our investigation, we utilized immortalized but non-transformed N-TERT-2G KCs (Dickson et al., 2000), which allowed us to select stable cell lines, thereby minimizing the experimental variability of transient infections, while the TET-inducible system allowed us to surmount the growth inhibitory effects of AREG knockdown. We also demonstrate that AREG is the most abundantly expressed EGFR ligand in N-TERT-KCs and show that NHK and N-TERT KCs display very similar levels of AREG shedding (Figure 2), which supports the use of immortalized N-TERT cells for the study of KC function.

Using the conditional shRNA expression system, we confirm our previous data with blocking Abs that AREG controls self-sustained proliferation and ERK phosphorylation of NHK (Stoll et al., 2010). We show that ERK phosphorylation is markedly reduced in response to AREG knockdown (Fig. 3) and that AREG shRNAs strongly inhibit KC proliferation and promote KC differentiation (Figs 4 and 5). Interestingly, ERK phosphorylation was more effectively blocked by anti-AREG Abs, whereas AREG shRNAs were much more inhibitory to KC growth (Figs 3-5). Furthermore, EGFR ligand treatment reversed the shRNA-induced effect on ERK phosphorylation (Fig. 3), but not the shRNA-mediated growth inhibition (Fig 5). These findings suggest that ERK phosphorylation by itself is not sufficient to sustain KC growth even though ERK activity is required for KC proliferation (Kim et al., 2008). Our data suggest an important function for KC-derived full-length AREG in this process.

It is still unclear why KC proliferation depends so strongly on AREG function. One obvious explanation is that KCs express far more AREG than any other EGF-like growth factor [(Stoll et al., 2010) and Figure 2]. However, this phenomenon could also involve altered trafficking of receptor-ligand complexes. AREG and EPGN were previously shown to have superior mitogenic activity compared with EGF or TGF-α, even though they are low affinity EGFR ligands (Adam et al., 1995; Kochupurakkal et al., 2005). It was suggested that their high mitogenic potential might be due to evasion of receptor-mediated endocytosis, which targets receptor-ligand complexes for intracellular degradation (Kochupurakkal et al., 2005). This would be consistent with the observation that addition of exogenous EGFR ligands did not improve N-TERT KC growth in the absence of TET (Fig. S1), suggesting that these cells produce sufficient levels of AREG to sustain growth. Surprisingly, we found that even high concentrations of recombinant EGFR ligands, up to 100 times the EGF concentration in regular KC growth media, could not rescue KC growth in AREG knockdown cells (Fig. 5). These results cannot be explained by a lack of activity of recombinant EGFR ligands because addition of EGF to cultures treated with anti-AREG restored cell growth to control levels and prevented the appearance of differentiated cells (Fig. 5). Moreover, addition of ligands to AREG knockdown cells induced ERK phoshorylation (Fig. 3) and profoundly changed cell morphology with the appearance of large, multinucleated KCs (Fig. 5).

In support of AREG as the predominant KC EGFR ligand mediating autocrine growth, our data demonstrate that AREG knockdown induces KC differentiation (Fig. 4). Interestingly, it was previously reported that inhibition of EGFR signaling in KCs leads to growth arrest, terminal differentiation and induction of keratins K1/10 (Peus et al., 1997; Poumay and Pittelkow, 1995). Taken together, our data further demonstrate that the endogenously expressed AREG TM precursor protein regulates KC growth and differentiation in a way that cannot be substituted by soluble EGFR ligands including AREG itself. This data suggest that in addition to mature, soluble AREG consisting of the EGF domain, other regions of the AREG TM precursor (possibly the carboxy-terminal domain) play an important role in the regulation of KC growth and differentiation. Interestingly, autocrine ERK phosphorylation (Fig. 3) and KC growth (Fig. 4A) were strongly blocked in the presence of the metalloproteinase inhibitor GM6001 (Stoll et al., 2010) further supporting published work (Sanderson et al., 2006) suggesting that proteolytic cleavage of the AREG TM precursor is required for proper function.

In support of an important function of the cytoplasmic domain for cell growth and differentiation, we show that expression of the AREG TM precursor in knockdown cells markedly restores KC proliferation (Figure 6), strongly reverses the morphological changes, and prevents the expression of KC differentiation markers (Fig. 4 and 6). However, it should be noted that the rescue was not complete. One possible explanation for this could be the observed TET-induced downregulation of EGFR (Fig. S2). Alternatively, it might be plausible that a Flag-Tag present at the carboxy-terminus of our construct interferes with its putative intracellular functions. Further studies are required to test these possibilities.

Interestingly, it was previously shown that the carboxy-terminal fragment of HB-EGF translocates to the nucleus where it binds to and inactivates promyelocytic zinc finger protein (PLZF), a transcriptional inhibitor of cyclin A (Nanba et al., 2003). Whether the C-terminal domain of AREG plays a similar role during cell cycle progression is unknown. However, the appearance of large, multinucleated cells in growth factor-treated AREG knockdown cells (Fig. 5) suggests that it might be important for cytokinesis. Recently, Isokane and colleagues demonstrated that TPA-induced AREG shedding results in endocytosis of both transmembrane-cytoplasmic AREG fragments and unshed transmembrane precursors in HeLa cells. It was shown that the AREG precursor translocates from the plasma membrane to the nucleus where it binds to lamin A, a nuclear envelope protein, leading to heterochromatinization and transient suppression of global gene transcription (Isokane et al., 2008). Interestingly, another study demonstrated that mutations in lamin A delay the onset and progression of cytokinesis leading to premature aging in dermal fibroblasts (Dechat et al., 2007). Further studies are needed to determine the relative importance of AREG and lamin A in controlling proliferation in skin KCs.

In summary, our data demonstrate that conditional, lentivirus-mediated gene knockdown is a versatile tool for regulating endogenous gene expression in non-transformed human KC. Using this tool, we show that autocrine human KC growth and differentiation depend on endogenously expressed AREG precursor protein, strongly suggesting a previously unreported function for the MP-generated carboxy-terminal domain of the AREG TM precursor. Further elucidation and targeting this function might be of therapeutic potential in inflammatory and neoplastic disorders of the skin and other epithelial tissues.

MATERIAL AND METHODS

Reagents

Recombinant human EGF was obtained from Peprotech (Rocky Hill, NJ). Recombinant AREG, HB-EGF and EPGN and antibodies against AREG, HB-EGF and TGF-α were from R&D Systems (Minneapolis, MN). The ErbB receptor tyrosine kinase inhibitor PD158780, the MEK inhibitor U0126, the MP inhibitor GM6001 and puromycin were purchased from Calbiochem (San Diego, CA). All other chemicals were obtained from Sigma (St. Louis MO).

Cell Culture

The immortalized, non-transformed human KC cell line N-TERT-2G, was provided by Dr. James Rheinwald (Boston, MA), and was routinely grown in KC serum-free medium (KSFM, Invitrogen) as previously described (Dickson et al., 2000). NHK and 293FT human embryonic kidney cells were cultured as described previously (Stoll et al., 2010).

Inducible and constitutive lentivirus-mediated RNA interference (RNAi) and gene expression

Small hairpin RNAs (shRNA) against AREG and EGFP were cloned into pLenti4/Block-iT-DEST (Invitrogen). The cDNA for EGFP was cloned into the TET-regulated lentiviral vector pLenti4/TO/V5-DEST (Invitrogen) as previously described (Stoll et al., 2010). Constructs encoding the AREG TM precursor with a c-terminal Flag-Tag or AREG ECD with a 3’ His-Tag (Figure 6A) were cloned into the lentiviral expression vector pLVX-aAcGFP-N1 (Clontech, Mountain View, CA) under the control of a constitutive CMV promotor. Lentiviral particles were produced according to the manufacturer's instruction (Invitrogen or Clontech) and used to stably transduce the TET repressor protein expressing cell line N-TERT-TR as described previously (Rittié et al., 2009; Stollet al., 2010).

Autocrine cell culture conditions

N-TERT KCs were plated at 5% confluence in complete KC serum-free medium (KSFM) and grown to 40-50% confluence. Cells were then incubated in basal M154 medium (Cascade Biologics, Portland, OR) or KSFM for 48 hours with or without 1 μg/ml TET.

Immunofluorescence

N-TERT-TR-EGFP cells were plated on glass coverslips and grown and treated as indicated in figure legends. The cells were fixed with 4% paraformaldehyde, counterstained with DAPI and photographed.

RNA isolation and quantitative RT-PCR

Total RNA was isolated and reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). Real-time PCR analysis was performed using TaqMan gene expression assays from Applied Biosystems as previously described (Rittié et al., 2009; Stoll et al., 2010). Target gene expression was normalized to the control gene 36B4 (RPLP0; Laborda, 1991; Minner and Poumay, 2009) and was expressed as % of untreated controls or as % of the control gene 36B4.

Cell Growth Assays

N-TERT KCs were plated at 1% confluence in complete KSFM and grown until the cell colonies contained approximately four to eight cells as previously described for NHK (Pittelkow et al., 1993). Cells were then incubated under autocrine growth conditions in basal KSFM medium for 6–8 days with and without 1μg/ml TET. Blocking anti-EGFR ligand or isotype control antibodies, with or without various concentrations of AREG, EGF, EPGN, HB-EGF and TGF-α were added to the cultures at the same time as TET. To assess KC cell growth, cells were washed with PBS, trypsinized and counted using a hemacytometer or stained with crystal violet and photographed.

Phospho-Erk western-blotting

N-TERT-2G cells were grown in KSFM medium to 40 to 50% confluence, followed by basal M154 medium for 48 hours. Incubation with inhibitors and western-blotting were performed as previously described (Stoll et al., 2010).

Enzyme-Linked ImmunoSorbent Assay (ELISA)

AREG protein levels in KC-conditioned medium was measured using an AREG DuoSet Elisa from R&D Systems as previously described (Kansra et al., 2004; Stoll et al., 2010).

Statistical Analysis

Data are expressed as mean +/- standard error of the mean (SEM). Statistical analyses were performed using paired or independent two-tailed Student's t-tests.

Supplementary Material

Supplement

ACKNOWLEDGMENTS

We thank Dr. James Rheinwald (Harvard Medical School, Boston, MA) for kindly providing the N-TERT-2G KCs. This work was supported by the National Institute for Arthritis, Musculoskeletal and Skin Disease (NIAMS), National Institutes of Health (NIH award K01 AR050462 and R03 AR049420 to SWS and R01 AR052889 to JTE). JTE is supported by the Ann Arbor Veterans Affairs Hospital.

Abbreviations used in this paper

Ab

antibody

AREG

amphiregulin

CMV

cytomegalovirus

ECD

extracellular domain

EGFR

epidermal growth factor receptor

ELISA

enzyme-linked immunosorbent assay

EPGN

epigen

ERK

extracellular signal-regulated kinase

GF

growth factor

HB-EGF

heparin-binding EGF-like growth factor

KC

keratinocyte

MP

metalloproteinase

NHK

normal human keratinocytes

QRT-PCR

quantitative real time polymerase chain reaction

RNAi

RNA interference

shRNA

small hairpin RNA

TET

tetracycline

TGF-α

transforming growth factor-α

TM

transmembrane precursor

Footnotes

CONFLICT OF INTEREST

The authors state no conflict of interest.

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