ADAM17 is the Principal Ectodomain Sheddase of the EGF-Receptor Ligand Amphiregulin

The epidermal growth factor (EGF)-receptor ligand amphiregulin (AREG) is a potent growth factor implicated in proliferative skin diseases and in primary and metastatic epithelial cancers. AREG in vitro, synthesized as a pro-peptide, requires conversion to an active peptide by metalloproteases by a process known as ectodomain shedding. Although, (Adam17) a disintegrin and metalloprotease 17 is implicated in ectodomain shedding of AREG, it remains to be established in vivo whether ADAM17 contributes to AREG shedding. In the present study, using a curly bare (Rhbdf2cub) mouse model that shows loss-of-hair, enlarged sebaceous glands, and rapid cutaneous wound-healing phenotypes mediated by enhanced Areg mRNA and protein levels, we sought to identify the principal ectodomain sheddase of AREG. To this end, we generated Rhbdf2cub mice lacking ADAM17 specifically in the skin and examined the above phenotypes of Rhbdf2cub mice. We find that ADAM17 deficiency in the skin of Rhbdf2cub mice restores a full hair coat, prevents sebaceous-gland enlargement, and impairs the rapid wound-healing phenotype observed in Rhbdf2cub mice. Furthermore, in vitro, stimulated shedding of AREG is abolished in Rhbdf2cub mouse embryonic keratinocytes lacking ADAM17. Thus, our data demonstrate that ADAM17 is the major ectodomain sheddase of AREG.


Introduction
The epidermal growth factor receptor (EGFR) pathway plays a major role in normal development, and in multiple diseases including epithelial cancers and chronic obstructive pulmonary disease, and in liver diseases [1][2][3][4][5]. A critical step in regulating this pathway is ectodomain shedding of type-1 transmembrane EGFR ligands from the cell surface by membrane-anchored metalloproteases [6]. For instance, type-1 transmembrane EGFR ligands, including amphiregulin (AREG), transforming growth factor alpha (TGFα), epidermal growth factor (EGF), and heparin-binding EGF (HB-EGF) are produced as inactive pro-peptides. In the ectodomain shedding process, ADAMs (a disintegrin and metalloproteases) cleave pro-peptides to release soluble peptides, leading to activation of the EGFR signaling pathway [7,8].
Furthermore, using loss-of-function experiments in mEFs, Sahin et al. demonstrated that both constitutive and stimulated ectodomain shedding of EGFR ligands, including EGF, epiregulin, betacellulin, HB-EGF, TGFA, and AREG, are unaltered in the absence of ADAM8, -9, -12, -15, and, -19 [9]. Thus, substantial literature suggests that ADAM10 and ADAM17 have essential, but distinct, roles in shedding of EGFR ligands. However, the specific metalloproteases contributing to the ectodomain shedding of AREG, a potent growth factor implicated in proliferative skin diseases, and primary and metastatic epithelial cancers [15][16][17], remain to be determined in vivo. Although results of in vitro studies have suggested ADAM17 as a key sheddase, ADAM8-, ADAM15-, and Batimastat (broad metalloprotease inhibitor)-sensitive metalloproteases have also been implicated in AREG shedding in vitro [11]. Understanding of the sheddase mechanisms for AREG is critical for development of more effective therapies for diseases associated with this growth factor.
To determine whether ADAM17 is the key sheddase of AREG, we utilized the curly bare (Rhbdf2 cub ) gainof-function mouse mutation. Homozygosity for this spontaneous mutation in the Rhbdf2 gene augments Areg mRNA and protein levels and results in alopecia, sebaceous-gland enlargement, and rapid-wound-healing phenotypes through enhanced secretion of AREG and subsequent hyperactivation of the EGFR pathway [18].
Furthermore, AREG deficiency in Rhbdf2 cub/cub mice prevents the alopecia, sebaceous gland enlargement, and rapid wound-healing phenotypes, suggesting that AREG is the primary mediator of the Rhbdf2 cub phenotype [18]. Thus, the Rhbdf2 cub mouse mutation provides a powerful in vivo model system that allows us to examine the physiological role of ADAM17 in ectodomain shedding of AREG and in AREG-mediated downstream events, including wound healing.
Here, we demonstrate that conditional deletion of ADAM17 in the skin of Rhbdf2 cub/cub mice impairs the AREG-mediated hair, sebaceous gland, and wound-healing phenotypes observed in these mice. We also demonstrate that ADAM17 deficiency significantly abolishes both stimulated and unstimulated shedding of AREG in Rhbdf2 cub/cub mouse embryonic keratinocytes (MEKs). Lastly, we show that enhanced shedding of AREG suppresses ADAM17 activity, suggesting that ADAM17 is a key ectodomain sheddase of AREG.

Materials and Methods
Animals. All animal work conformed to regulations in the Guide for the Care And Use of Laboratory Animals Histology. Mice were euthanized by CO 2 asphyxiation followed by open chest necropsy, a secondary method of euthanasia. Dorsal skin was removed, fixed in 10% neutral buffered formalin for 24 hours, processed routinely, embedded in paraffin, sectioned and stained, with hematoxylin and eosin (H&E).
Isolation of primary keratinocytes. For isolation of mouse embryonic keratinocytes (MEKs), skin from embryonic day 18 mouse embryos was incubated overnight in Neutral Protease at 4°C. Following separation of the epidermis from the dermis, the epidermis was placed in Petri dishes containing trypsin (# 12563029,ThermoFisher Scientific) and allowed to incubate for 30 min at room temperature. After blocking trypsin activity with soybean trypsin inhibitor (# R007100, ThermoFisher Scientific), cells were grown in KBM-2 medium (# CC-3107, Lonza) supplemented with antibiotic/antimycotic.

Measurement of amphiregulin protein levels.
AREG levels in the cell culture supernatant were measured via ELISA as described previously [18]. Briefly, 100 µL of cell-culture supernatant was added to capture antibody-pre-coated plates and incubated for 2 h at room temperature (RT). After three washes, 100 µL of the detection antibody was added to each well and incubated for an additional 2 h at RT. Following three washes, 100 µL of Streptavidin-HRP was added to each well and incubated at RT for 20 min, before adding 100 µL of substrate solution (20 min incubation) and 50 µL of stop solution. A spectrophotometer (SpectraMax 190, Molecular Devices) was used to determine the optical density.
Dextran sulfate sodium (DSS)-induced colitis. Rhbdf2 +/+ , Rhbdf2 cub/cub , and Rhbdf2 cub/cub Areg -/mice were exposed to 2% DSS (MP Biomedicals) for 7 days via drinking water and their body weights were recorded each day for 10 days. The percentage of change in body weight for each mouse strain was documented daily and the differences between the strains were analyzed. After 10 days, mice were euthanized and their colons were examined for hallmarks of colitis.

SDS/PAGE and immunoblotting.
These assays were performed as described previously (10). Briefly, MEFs grown in 6-well dishes were lysed with RIPA buffer (Cell Signaling, MA), and protein concentrations were determined using a Qubit Fluorometer (Life Technologies). After loading 35 µg of protein onto 4-20% (wt/vol) precast gels (Lonza), proteins were transferred to a PVDF membrane followed by blocking with 5% milk for 1 hr at RT. Membranes were then exposed to ADAM17-specific antibody (1:1000, ab2051, Abcam, MA) for 2 hr at RT. Subsequently, membranes were washed in TBST for 2 hr, exposed to secondary antibody (1:5,000, Santa Cruz Biotechnology) for 1 hr at RT, and washed for 2 hr in TBST. Membranes were then exposed to Luminol reagent (Santa Cruz Biotechnology) for 3 min and visualized using a gel doc system (G:BOX F3, Syngene).
Statistical analysis. One-way ANOVA was used for comparison of several groups using Prism v7 software (GraphPad). A p-value < 0.05 was considered statistically significant. Data represent mean ± SD.

Results
The hair-loss, sebaceous-gland enlargement, and rapid wound-healing phenotypes of Rhbdf2 cub mice are mediated through ADAM17 To determine whether ADAM17 is essential for the hair-loss and enlarged sebaceous gland phenotypes exhibited by Rhbdf2 cub/cub mice, we generated Rhbdf2 cub/cub mice lacking ADAM17 in skin, by crossing Rhbdf2 cub/cub mice with Adam17 flox/flox K14-Cre mice, and studied the phenotypes of second-generation offspring. We noted that ADAM17 acts as a genetic modifier of Rhbdf2 cub/cub mice-Rhbdf2 cub/cub Adam17 flox/flox K14-Cre mice display a full hair coat, in contrast to the complete hair loss in Rhbdf2 cub/cub mice (Fig. 1A). We next performed histopathological examination of truncal skin from Rhbdf2 cub/cub (Fig. 1B, a and b) and We next examined the rapid wound-healing phenotype of Rhbdf2 cub/cub mice. The Rhbdf2 cub mutation induces a rapid wound-healing phenotype through enhanced secretion of AREG [18,20]; when we punched 2mm through-and-through holes in the ear pinnae of Rhbdf2 cub/cub mice, within two weeks ear-hole closure of more than 90% was observed in Rhbdf2 cub/cub mice, in contrast to approximately 20% ear-hole closure in Rhbdf2 +/+ mice [18]. Here, we wanted to determine whether the wound-healing phenotype in Rhbdf2 cub/cub mice requires ADAM17. Using the above-mentioned ear-hole closure assay, we tested the wound-healing phenotype of Rhbdf2 cub/cub Adam17 flox/flox K14-Cre mice and compared it to those of Adam17 flox/flox K14-Cre and Rhbdf2 cub/cub mice. Not surprisingly, impairment of wound healing was similar in Adam17 flox/flox K14-Cre mice ( Fig. 1C, left column) and Rhbdf2 cub/cub Adam17 flox/flox K14-Cre mice (Fig. 1C, middle column), whereas Rhbdf2 cub/cub mice showed the rapid wound-healing phenotype (Fig. 1C, right column). Collectively, these data suggest that loss of ADAM17 in the skin of Rhbdf2 cub/cub mice modifies the hair-loss phenotype and restores a full hair coat, and diminishes the wound-healing phenotype.
Next, using flow cytometry analyses we determined whether there was any indication of myeloproliferative disease in Rhbdf2 cub/cub Adam17 flox/flox K14-Cre mice by quantifying the differences in the percentages of splenic macrophages (Fig. 2C, top panel) and neutrophils (Fig. 2C, bottom panel) between ADAM17 flox/flox K14-Cre and Rhbdf2 cub/cub ADAM17 flox/flox K14-Cre mice. Compared with control mice (ADAM17 flox/flox and ADAM17 flox/+ mice), we observed significantly higher percentages of macrophages and neutrophils in both of ADAM17 flox/flox K14-Cre and Rhbdf2 cub/cub Adam17 flox/flox K14-Cre mice, suggesting that loss of ADAM17 specifically in the skin results in considerable myeloproliferation in Rhbdf2 cub/cub mice. Taken together, our results indicate that lack of ADAM17 in the skin results in dermatitis and myeloproliferative disease, which validates previous findings by Franzke et al. that ADAM17 maintains the skin barrier [19]. Moreover, our results showing development of a similar overt skin phenotype observed by Franzke et al. and restoration of hair growth in Rhbdf2 cub/cub mice lacking ADAM17 implicate ADAM17 as a participant in ectodomain shedding of AREG.
Fourth, similar results were obtained when keratinocytes isolated from the aforementioned strains (1 through 6) of mice were exposed to bacterial endotoxin lipopolysaccharide (LPS) and assayed for AREG levels in the culture supernatants (Fig. 3B). Interestingly, there was a subtle but significant increase in the levels of AREG in Adam17 flox/flox K14-Cre and Rhbdf2 cub/cub Adam17 flox/flox K14-Cre keratinocytes upon stimulation with LPS ( Fig. 3B; 5 and 6); however, this could be due to differential regulation of AREG constitutive shedding by PMA versus LPS. Taken together, because loss of ADAM17 significantly abolished stimulated secretion of AREG in Rhbdf2 cub/cub keratinocytes, these data strongly suggest that ADAM17 is essential for ectodomain shedding of AREG (Fig. 3C).

Enhanced shedding of substrate suppresses ADAM17 sheddase activity
Both our group (Hosur et al., 2014) and Siggs et al. (Siggs et al., 2014) recently observed that ADAM17 activity is reduced in Rhbdf2 cub/cub mice. We sought to validate these findings by assessing the susceptibility of Rhbdf2 cub/cub mice to DSS-induced colitis. Increased susceptibility to DSS-induced colitis is an indicator of reduced ADAM17 activity; conditional ADAM17 knockout mice and ADAM17 hypomorphic mice are highly susceptible to DSS-induced colitis because of impaired EGFR signaling [21,22]. To examine whether Rhbdf2 cub/cub mice are hypomorphic for ADAM17, in which case they would be more susceptible than control mice to DSS-induced colitis, we provided mice with 2% DSS in drinking water for seven days, followed by water for three days, and recorded the body weight and percentage of change in body weight of Rhbdf2 +/+ and Rhbdf2 cub/cub mice. While both Rhbdf2 +/+ and Rhbdf2 cub/cub mice experienced weight loss during the seven-day DSS treatment period, Rhbdf2 cub/cub mice continued to experience weight loss during the three-day post-DSS treatment period, while the weight of Rhbdf2 +/+ mice stabilized during that period (Fig. 4A). At the end of the ten-day experimental period, Rhbdf2 cub/cub mice had lost significantly more body weight than Rhbdf2 +/+ mice ( Fig. 4A), suggesting that Rhbdf2 cub/cub mice are more susceptible to DSS-induced colitis. At the conclusion of the ten-day DSS test, experimental animals were euthanized, their colons were removed, and the colonic lengths were compared (Fig. 4B). The colons of Rhbdf2 +/+ mice were significantly longer than those of Rhbdf2 cub/cub mice after DSS exposure, further indicating that Rhbdf2 cub/cub mice are more susceptible to DSSinduced colitis. Thus, these data show that ADAM17 activity is significantly reduced in Rhbdf2 cub/cub mice.

Discussion
AREG plays an important role in pathological processes, including psoriasis induction [23,24], cancer progression, and resistance to chemotherapy and anti-EGFR therapies [15,25]. For example, AREG has been characterized as a multicrine-autocrine, paracrine, and endocrine (systemic)-growth factor in primary and metastatic epithelial cancers [26][27][28]. AREG induces its own expression to enable self-sufficiency of growth signals acting through EGFR, via an extracellular autocrine loop [29], suggesting that dysregulation of this loop could lead to overexpression of AREG. Additionally, cancer cells overexpressing AREG can induce neoplastic transformation of neighboring cells through paracrine or endocrine activity [30]. Also, more recently, we showed in mice that AREG underlies the hyperproliferative skin disease tylosis, and that loss of AREG restores the normal skin phenotype in a mouse model of human tylosis [31]. Together, these studies highlight the key role of AREG in several pathological processes, and the potential of AREG depletion as a therapeutic approach in multiple diseases. To develop effective therapeutic strategies targeting AREG, it is important to understand how AREG secretion is regulated in vivo.
AREG synthesized as pro-AREG is converted to an active form by metalloproteases. Although several ADAMs have been implicated, a study by Sahin et al. [9] showed that Adam17 -/-MEFs exhibit impaired shedding of AREG, indicating that ADAM17 may be a major sheddase. Comparison of the phenotype of Adam17 -/mice with that of Areg -/mice is a potential means of providing support for a role of ADAM17 as the major sheddase of AREG. However, literature suggests that in contrast to Hbegf -/and Tgfa -/mice [13,14], Areg -/mice are viable and do not present with an overt phenotype, except for defects in mammary gland development during puberty and in nursing [32]. Thus, it remains to be determined whether the phenotype of mice with Areg depletion resembles any aspects of the Adam17 -/phenotype. Adam17 -/mice exhibit perinatal lethality, limiting the ability to examine mammary gland development and nursing competence phenotypes.
Furthermore, although at birth Adam17 -/pups exhibit stunted growth and development [33], including defective mammary branching, suggesting a role for ADAM17 in shedding of AREG, there is a lack of direct evidence. In the present study, using mouse genetics and in vitro ectodomain shedding assays, we sought to determine whether loss of ADAM17 abolishes shedding of AREG in vivo. We demonstrate that loss of ADAM17 impairs the AREG-mediated loss-of-hair, sebaceous-gland enlargement, and rapid wound-healing phenotypes observed in Rhbdf2 cub/cub mice. Moreover, we find that conditional deletion of ADAM17 in the skin of Rhbdf2 cub/cub mice significantly inhibits stimulated secretion of AREG in keratinocytes. Together, these results suggest that ADAM17 is essential for ectodomain shedding of AREG. wrote the paper.

Conflict of Interest Statement
The authors declare that no conflict of interest exists.   A. ELISA quantitation of cleaved AREG from the supernatant of MEKs after overnight stimulation with either DMSO or 100 nM PMA. ND, not detected; Data represent mean ± SD. B. ELISA quantitation of cleaved AREG from the supernatant of MEKs after overnight stimulation with either PBS or 1 µg/mL LPS. ND, not detected; Data represent mean ± SD. C. Loss of ADAM17 modifies the Rhbdf2 cub/cub hair-loss and ear-punch-closure phenotypes and in vitro deficiency of ADAM17 in Rhbdf2 cub/cub keratinocytes prevents stimulated-secretion of AREG, together suggesting that ADAM17 is indispensible for sheddase of AREG. A. Percentage of change in body weights of 8-week-old female Rhbdf2 +/+ and Rhbdf2 cub/cub mice over a period of 7 days of exposure to DSS followed by 3 days of water; ***p<0.001. B. Colon lengths from control Rhbdf2 +/+ (n=1) and Rhbdf2 cub/cub mice (n=2) on water, and Rhbdf2 +/+ (n=4) and Rhbdf2 cub/cub (n=4) mice on DSS, at the end of the experimental period; ***p<0.001. C. Percentage of change in body weights of Rhbdf2 +/+ Areg +/+ and Rhbdf2 cub/cub Areg -/mice over a period of 7 days of exposure to DSS followed by 3 days of water. D. Immunoblot analysis of cell lysates obtained from Rhbdf2 +/+ , Rhbdf2 cub/cub , and Rhbdf2 cub/cub Areg -/-MEFs before and after stimulation with 100 nM PMA for 24 h. Top row = before stimulation; middle row = after stimulation. Actin served as loading control. pro, pro-ADAM17; m, mature ADAM17. See Supplemental Figure 1 for full blots. E. The observation that ADAM17 activity is significantly reduced in Rhbdf2 cub/cub mice indicates that enhanced shedding of AREG may lead to downregulation or internalization of ADAM17.
Supplemental Figure 1