Dysregulation of ADAM10 shedding activity in naked mole‐rat fibroblasts is due to deficient phosphatidylserine externalization

The naked mole‐rat (NMR, Heterocephalus glaber) is of significant interest to biogerontological research, rarely developing age‐associated diseases, such as cancer. The transmembrane glycoprotein CD44 is upregulated in certain cancers and CD44 cleavage by a disintegrin and metalloproteinase 10 (ADAM10) regulates cellular migration. Here we provide evidence that mature ADAM10 is expressed in NMR primary skin fibroblasts (NPSF), and that ionomycin increases cell surface ADAM10 localization. However, we observed an absence of ADAM10 mediated CD44 cleavage, as well as shedding of exogenous and overexpressed betacellulin in NPSF, whereas in mouse primary skin fibroblasts ionomycin induced ADAM10‐dependent cleavage of both CD44 and betacellulin. Overexpressing a hyperactive form of the Ca2+‐dependent phospholipid scramblase ANO6 in NPSF increased phosphatidylserine (PS) externalization, which rescued the ADAM10 sheddase activity and promoted cell migration in NPSF in an ADAM10‐dependent manner. These findings suggest that dysregulation of ADAM10 shedding activity is due to a deficient PS externalization in NMR.

. In addition, CD44 has been associated with cancer progression, being one of the most consistent markers of cancer stem cells and is involved in their generation, maintenance, and survival (Hassn Mesrati et al., 2021;Leung et al., 2010;Lokeshwar et al., 1995;Takaishi et al., 2009).
The principal domains of CD44 are the extracellular domain or ectodomain, the transmembrane domain, and the intracellular/ cytoplasmic domain (Lesley and Hyman, 1998). The CD44 ectodomain can be cleaved by metalloproteases (Nagano, Murakami et al., 2004;Nakamura et al., 2004), a process stimulated by extracellular Ca 2+ influx, the activation of Rho family small GTPases, Rac and Ras oncoproteins, and the activation of protein kinase C (Kawano et al., 2000;Nagano, Murakami et al., 2004;. Cleavage of the CD44 ectodomain is highly prevalent in patients with tumors including glioma, breast, lung, colon, and ovarian cancers (Okamoto et al., 2002;Yamane et al., 1999), and high levels of soluble CD44 have been found in the serum of patients with cancer (Y. J. Guo et al., 1994;Masson et al., 1999). Furthermore, CD44 cleavage promotes tumor cell migration (Kawano et al., 2000;Kolliopoulos et al., 2021;Kung et al., 2012;Sugahara et al., 2003). Therefore, it is clear that CD44 cleavage plays an important role in tumor progression.
A disintegrin and metalloprotease 10 (ADAM10) is one of the metalloproteases involved in CD44 cleavage (Anderegg et al., 2009;Murai et al., 2006). ADAM proteins are type I metalloproteases responsible for the shedding of different membrane-bound receptors and ligands controlling many cellular functions (Reiss & Saftig, 2009).
ADAM10 is one of best characterized ADAM family members, cleaving many proteins, including CD44, betacellulin (BTC), and Notch (Hartmann et al., 2002;Nagano, Murakami et al., 2004;Sahin et al., 2004), as well as being involved in diverse physiological processes, such as fertilization, neurogenesis, and angiogenesis Sahin et al., 2004). ADAM10 dysregulation is associated with different pathologies, such as embryonic lethality in mice due to disturbed Notch signaling being caused by depletion of the ADAM10 gene (Hartmann et al., 2002) and upregulation of ADAM10 occurring in melanoma (Lee et al., 2010), as well as roles for dysregulation of ADAM10 shedding activity occurring in Alzheimer's disease, and cancer development (Marcello et al., 2017;Murphy, 2008). Constitutive ADAM10 shedding activity is enhanced by Ca 2+ influx, which can be induced by ionophores as ionomycin (IM) Reiss & Bhakdi, 2012), as well as exposure of the negatively charged phosphatidylserine (PS) in the outer leaflet of the cell membrane (Bleibaum et al., 2019).
Considering the role of ADAM10 in cellular function, we hypothesize that another mechanism that might contribute to NMR cancer resistance would be dysregulation of ADAM10 shedding activity. In experiments conducted in NMR primary skin fibroblasts (NPSF), we report an absence of CD44 cleavage induced by IM.
Furthermore, although IM induced translocation of ADAM10 to the cell membrane of NPSF, it does not induce ADAM10-dependent shedding of the ADAM10 substrate BTC when overexpressed. However, when NPSF overexpressed a hyperactive form of anoctamin 6 (ANO6-HA, a Ca 2+ -dependent phospholipid scramblase), an increase in PS externalization was observed alongside an IM induced, ADAM10-dependent BTC shedding. Finally, ANO6-HA overexpression promoted ADAM10-dependent cell migration. All these data suggest that a deficiency in PS externalization may contribute to a dysregulation of ADAM10 shedding activity in NPSF.

| Animals
Experiments were performed on cells isolated from a mixture of male and female C57BL6/J mice (8−15 weeks old), and a mixture of male and female, nonbreeder NMRs (7−43 months old). Mice were conventionally housed with nesting material and a red plastic shelter in temperature-controlled rooms at 21°C, with a 12 h light/dark cycle and access to food and water ad libitum. NMRs were bred in-house and maintained in an interconnected network of cages in a humidified (~55%) temperature-controlled room at~28°C, with red lighting 2.2 | NMR and mouse primary skin fibroblast isolation NMR and mouse primary skin fibroblast (NPSF and MPSF) isolation were performed as described before (Hadi et al., 2020). Briefly, NMR were humanely killed by CO 2 exposure followed by decapitation, whereas mice were killed by cervical dislocation of the neck and cessation of circulation. Skin tissue was collected from each animal on ice-cold PBS 1X (#70011-044; Gibco). For NPSF, skin was taken from the underarm, dorsal, and ventral surfaces of each animal, and was cleaned of any fat or muscle tissue and sprayed with 70% ethanol. For MPSF, both external ears were collected from each animal and was cleaned of any hair and sprayed with 70% ethanol.
The mouse pinna lacks significant hair, like NMR skin in general, but NMR do not have pinnae and hence it was not feasible to isolate fibroblasts from glabrous skin of identical origins in both species; NPSF grow slowly and thus isolating from multiple regions enabled experiments to be conducted in a timely manner, as well as minimizing the number of animals used, and when tracking the area from where fibroblasts were isolated no difference between experiments was found. Once isolated and cleaned, tissue was washed twice with PBS 1X and finely minced with sterile blades (#BS2982; Swann Morton). For dissociation, minced skin was incubated at 37°C for 3-5 h in 5 mL of DMEM high glucose (#41965-039; Gibco) supplemented with 10 mg mL −1 collagenase (#C9891; Sigma-Aldrich), 1000 units mL −1 hyaluronidase (#H3506; Sigma-Aldrich). The tissue was briefly vortexed every 30 min. Cells were subsequently pelleted by centrifuging at 500g for 3 min and resuspended in PBS 1X, then the cells were centrifuged again and resuspended in cell culture medium: DMEM high glucose (#41965-039; Gibco) supplemented with 15% fetal bovine serum (#F7524-500ML; Sigma-Aldrich), 100 units mL −1 penicillin, 100 μg mL −1 streptomycin (#15140122; Gibco), and 100 μg mL −1 Primocin (#antpm-2; InvivoGen). NMR culture media was further supplemented with 1X non-essential amino acids (#11140-050; Gibco) and 1 mM sodium pyruvate (#11360-039; Gibco). This cell suspension was passed through a 70 μm filter (#352350; Falcon) and seeded on a treated cell culture flask (T-75 #658175; Greiner Bio-One). NPSF cultures were incubated in a humidified 32°C incubator with 5% CO 2 and 3% O 2 , whereas MPSF cultures were incubated in a humidified 37°C incubator with 5% CO 2 ; as stated in the Results, in some experiments, MPSF were cultured at 3% O 2 and 32°C, cells being exposed to these conditions for seeding, transfection, and the experiment itself.

| Protein extraction and western blot analysis
images were analyzed using the Image Studio Lite software (LI-COR).

| Membrane protein biotinylation
Fibroblasts were grown in a 10 cm dish (#CC7682-3394; Cyto-one). Hanks-Ca 2+ supplemented with 1 mg mL −1 glycine. Subsequently, cells were recollected in a solution of distilled water containing a protease inhibitor cocktail as described above (#P2714; Sigma-Aldrich) and centrifuged at 16,100 × g for 2 min at 4°C. The pellet was resuspended in a buffer containing 150 mM NaCl, 10 mg/mL PMSF, 60 mM DTT, 1% NP-40, 1 mM sodium orthovanadate, and 50 mM Tris-HCl pH 7.4, plus a general metalloprotease inhibitor BB-94 10 µM (#196440; Calbiochem) and protease inhibitor cocktail as described above (#P2714; Sigma-Aldrich). The protein concentration (total fraction) was calculated, and 200 µg of protein was precipitated with NeutrAvidin™ Agarose Resins (#29201; Thermo Fisher Scientific) after an incubation of 1 h at 4°C with occasional manual agitation. The pellet was washed with Hanks without Ca 2+ supplemented with 0.1% SDS and 1% NP-40 and then centrifuged at 16,100 × g for 2 min at 4°C and resuspended with 20 µL Hanks-Ca 2+ supplemented with 1 mg mL −1 glycine, pH 2.8. Then the sample was centrifuged in the same conditions above and the supernatant was rescued (this was the biotinylated fraction). All fractions were run on a 10% polyacrylamide gel (SDS-PAGE) under reducing and denaturing conditions.
The localization of ADAM10 was assayed in fibroblasts treated for 1 h with IM 0.5 µM or DMSO as vehicle. Fibroblasts were fixed in 100% methanol for 15 min at −20°C and then permeabilized with 0.1% Triton X-100. Samples were blocked with 3% BSA (#A2153; Sigma-Aldrich) in TBS containing 0.1% Tween-20 (T-TBS) for 1 h at room temperature. A primary antibody against ADAM10 (#PA5-87899; Invitrogen; host-rabbit) was applied at 1 μg mL −1 diluted in bocking solution and dishes were incubated overnight at 4°C in a humidified chamber. The next day, the dish was washed three times for 10 min in T-TBS. Goat anti-rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488 (#A-11008; Invitrogen) at 1 μg mL −1 diluted in 3% BSA-T-TBS was added to the dish and then incubated for 1 h at room temperature, before washing dishes as before. Subsequently, 5 µg mL −1 of wheat germ agglutinin was added and incubated for 10 min at 37°C. Then the dishes were washed as before and NucRed™ Live 647 (#R37106; Invitrogen) was added to the dish and incubated for 5 min at room temperature. Finally, dishes were washed as above and cells observed using a Leica SP5 laserscanning confocal microscope with x63 oil objective. Images were acquired in sequential scan mode: wheat germ agglutinin fluorescence was excited with a 405 nm laser and emission 415−475 nm recorded, Alexa Fluor™ 488 was excited with a 488 nm laser and emission between 498 and 590 nm captured, a 633 nm laser was used to excite NucRed™ Live 647 and emission between 645 and 633 nm detected. Composite images were created in ImageJ.

| Cell transfection
Mouse and NMR primary and SV40-Ras skin fibroblasts were transfected with plasmids encoding alkaline phosphatase (AP)tagged BTC (Horiuchi et al., 2007) or with the hyperactive ANO6 (ANO6-HA) (Bleibaum et al., 2019). Briefly, fibroblasts were seeded to 80% confluence and the following day were washed with PBS 1X and cultured in DMEM high glucose (#41965-039; Gibco) using the same conditions described above. Cells were cultured for 24 h with a complex of DNA and FuGENE ® HD Transfection Reagent (#E2311; Promega) at a ratio of 1:3 prepared in Opti-MEM reduced serum medium (#31985070; Gibco). Tris, 100 mM NaCl, 20 mM MgCl 2 , pH 9.5) was added only to the cell lysate wells. After that, both conditional media and cell lysate, were incubated with 100 µL of 2 mg mL −1 p-NPP (p-nitrophenyl phosphate) substrate (#34045; Thermo Fisher Scientific) diluted in APbuffer. Then the plate was incubated for 1 h at 37°C. The AP activity was measured by a spectrophotometer (CLARIOstar; BMG Labtech) at 405 nm. Three identical wells were prepared, and the ratio of AP activity in the medium and that of the cell lysate plus medium was calculated. For stimulated shedding or vehicle, the fold increase in the ratio of AP activity obtained after stimulation is shown relative to ratio of AP activity in control wells. Each experiment was conducted at least three times. Transfection efficiency was controlled by determining the ratio of the activity of AP in the medium over the AP-activity in the cells plus the medium.

| PS quantification in the outer leaflet of the cell membrane
Twenty thousand fibroblasts per well were seeded in a white 96 well plate (#CC7682-7596; CytoOne). The following day, cells were washed with PBS 1X and incubated with Imaging media (140 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2 , 20 mM HEPES) supplemented with 15% of fetal bovine serum (#F7524-500ML; Sigma-Aldrich). PS exposure was evaluated through the ratio between the Annexin V and number of cells. Annexin V binding to PS was measured using the RealTime-Glo™ Annexin V luminescence assay (#JA1000; Promega), following the manufacturer's instructions, binding of PS to annexin V generating a luminescence signal. The relative luminescence unit (RLU) was measured using a luminescence microplate reader (CLARIOstar; BMG Labtech). To determine the number of cells, after measurement of the RLU, cells were washed once with PBS 1X and then incubated with NucRed™ Live 647 ReadyProbes ® reagent (Invitrogen #R37106) in Imaging media for 15 min at room temperature. Cells were then washed with PBS 1X and the relative fluorescence units (RFU) was measured using a fluorescence microplate reader (CLARIOstar; BMG Labtech). Three identical wells were prepared, and the ratio of RLU/RFU was calculated.

| Wound closure assay
Twenty thousand fibroblasts were plated in each well of a cultureinsert 2 well in µ-dish 35 mm, high (#81176; Ibidi) and transfected as mentioned above. The following day, the silicon insert was removed from the dish and cells were washed once with PBS 1X and DMEM high glucose media (#41965-039; Gibco), supplemented with 5% of fetal bovine serum (#F7524-500ML; Sigma-Aldrich). During all the experiments, fibroblasts were culture in the conditions mentioned above. To measure the wound closure percentage, four pictures from each dish were taken at 0, 3, 6, 9, 12, 24, 36, and 48 h using a 5MP USB 2.0 Color CMOS C-Mount Microscope Camera (MU500-CK-3PL; AmScope) and the wound closure area was calculated using the TScratch software (Gebäck et al., 2009).
We therefore used immunoblotting to analyze the extent of IMinduced CD44 cleavage by ADAM10 in NPSF and mouse primary skin fibroblasts (MPSF); IM is a well characterized initiator of Ca 2+ influx that activates ADAM10 sheddase activity .
In all conditions, cells were preincubated for 30 min with the proteasomal inhibitor MG-132 (10 µM) to prevent degradation of CD44 cleavage products (Okamoto et al., 2001). In MPSF, the CD44 full length protein had a molecular weight of~100 kDa, comparable to that previously published (Kolliopoulos et al., 2021;Okamoto, Kawano, Tsuiki et al., 1999). After 1 h treatment with 0.5 and 1 µM IM, increased density of two fragments between~15 and~25 kDa was observed in MPSF (Supporting Information: Figure S1A). To determine the participation of metalloproteases, and specifically ADAM10 in this process, the effects of the following drugs were observed: batimastat, a general metalloprotease inhibitor (BB-94, 10 µM), GW280264X, an inhibitor of ADAM10/ADAM17 (GW, 1 µM), and GI254023X, which is selective for ADAM10 (GI, 1 µM) (A. Ludwig et al., 2005). In MPSF, all three inhibitors prevented IM-induced CD44 cleavage (Figure 1a), suggesting a key role for ADAM10 in CD44 cleavage in MPSF. By contrast, in NPSF, IM failed to induce CD44 cleavage using the same IM concentrations and incubation times that induce CD44 cleavage in MPSF (Figure 1b and Supporting Information: S1B). These data show that CD44 cleavage is not induced by IM in NPSF.

| NPSF express ADAM10, which undergoes cell membrane translocation, but is not activated by IM
To determine if the lack of IM-induced CD44 cleavage in NPSF is due to lack of ADAM10 expression, we compared ADAM10 expression levels in NPSF and MPSF. Pro-and mature ADAM10 were observed in both MPSF and NPSF, higher levels of pro-ADAM10, but not mature ADAM10, being expressed in NPSF compared to MPSF

| IM induces CD44 cleavage and ADAM10 shedding activity in both mouse and NMR SV40-Ras skin fibroblasts
To determine if the absence of IM-induced ADAM10 shedding activity is a general phenomenon of NMR cells, or is restricted to primary cells, we conducted experiments using transformed mouse and NMR SV40-Ras skin fibroblasts previously developed in our lab (Hadi et al., 2020). First, IM-induced CD44 cleaved protein levels were determined by immunoblot. In both mouse and NMR SV40-Ras skin fibroblasts, 0.5 µM IM for 1 h did not induce an increase of CD44 cleavage products (data not shown). Therefore, we evaluated if CD44 cleavage could be induced by 2.5 µM IM, a concentration previously reported to induce ADAM10 shedding activity in mouse embryonic fibroblasts (Urriola-Muñoz et al., 2018), at 5, 10, 15, and 30 min. A significant increase of two fragments between~15 and~25 kDa was observed after 15 min treatment with 2.5 µM IM in mouse cells (Supporting Information: Figure S2A) and after 10 min treatment in NMR cells (Supporting Information: Figure S2B). In SV40-Ras skin fibroblasts of both species, CD44 cleavage was prevented by batimastat (BB-94, 10 µM), GI254023X (GI, 1 µM), or GW280264X (GW, 1 µM) (Figure 3a,b), suggesting that ADAM10 participates in IM-induced CD44 cleavage in both mouse and NMR SV40-Ras cells.
In addition, pro-and mature ADAM10 is expressed in both, mouse and NMR SV40-Ras skin fibroblasts, with higher pro-ADAM10, but not mature ADAM10, levels in mouse compared to NMR cells (Supporting Information: Figure S3A). An IM-induced increase in mature ADAM10 was found in the biotinylated cell membrane protein fraction of both mouse and NMR SV40-Ras skin fibroblasts, with no change detected in the total fraction (Supporting Information: Figure S3B). Similar to results obtained when measuring IMinduced CD44 cleavage, a significant increase in the shedding of BTC URRIOLA-MUÑOZ ET AL. | 765 after incubation with 2.5 µM IM was observed in both mouse and NMR SV40-Ras skin fibroblasts transfected with the AP-tagged ADAM10 substrate BTC. This increase was prevented with the batimastat (BB-94, 10 µM), GI254023X (GI, 1 µM), or GW280264X (GW, 1 µM) (Figure 3c,d). Overall, these data suggest that IM stimulated ADAM10 activity, inducing ADAM10-dependent CD44 cleavage and BTC shedding in transformed cells from both mouse and NMR, thus demonstrating that NMR ADAM10 is functional in certain conditions.

| NMR SV40-Ras skin fibroblasts have more PS in the outer leaflet of the cell membrane
To try to elucidate why IM fails to induce ADAM10 shedding activity in NPSF, but does in NMR SV40-Ras skin fibroblasts, the localization of the phospholipid PS in the outer leaflet of the cell membrane was evaluated. This is because surface exposure of PS on the cell membrane has been proposed to regulate ADAM10 shedding activity (Bleibaum et al., 2019). Using the RealTime-Glo™ Annexin V F I G U R E 1 CD44 cleavage is not induced by ionomycin (IM) in NPSF. An immunoblot was performed to evaluate the full length and cleaved products of CD44 in (a) MPSF and (b) NPSF. Cells were treated for 30 min with a general inhibitor of metalloproteases (batimastat, BB, 10 µM), an ADAM10 inhibitor (GI254023X, GI, 1 µM), or an ADAM10/ADAM17 inhibitor (GW280264X, GW, 1 µM), before stimulating cells for 1 h with IM (0.5 µM). (a) In MPSF, the full CD44 protein has a molecular weight of~100 kDa, when CD44 is cleaved, two fragments between~15 and 25 kDa are produced. Comparison of the upper and lower cleavage products with the full protein level generated a ratio of CD44 cleavage/total. (b) In NPSF, the full CD44 protein has a molecular weight of~100 kDa, when CD44 is cleaved, only one fragment of~15 kDa was observed. Comparison of the cleavage product with the full protein level generated a ratio of CD44 cleavage/total. Vehicle: DMSO. Results are mean ± SEM; n = 3. For statistical analysis mean values were compared using an ANOVA and Tukey's post hoc test; *p ≤ 0.05. ADAM10, A disintegrin and metalloprotease 10; MPSF, mouse primary skin fibroblasts; NPSF, NMR primary skin fibroblasts. luminescence assay, the binding of annexin V with PS in the outer leaflet of the cell membrane was measured, providing a readout in RLU. Simultaneous staining of the nucleus with the NucRed™ Live 647 ReadyProbes ® reagent provided a readout of RFU, indicative of the number of cells. The ratio of both parameters was determined in MPSF, NPSF, mouse, and NMR SV40-Ras skin fibroblasts (Figure 4a).
No differences were found in the PS exposure to the outer leaflet in MPSF versus mouse SV40-Ras cells, but an approximately 10-fold higher level of PS was found in MPSF compared to NPSF. Moreover, a threefold higher level of PS was found in NMR SV40-Ras cells compared to NPSF (Figure 4b). This difference in PS levels could explain why IM-induced ADAM10 shedding was observed in NMR SV40-Ras, but not NPSF.

| Overexpression of ANO6-HA rescues IM-induced ADAM10 shedding activity in NPSFs
To test whether increasing PS in the outer leaflet of the cell membrane could enhance IM-induced ADAM10 shedding activity, MPSF and NPSF were transfected with either a hyperactive form of anoctamin 6 (ANO6-HA) or GFP as a control. ANO6, is a Ca 2+dependent phospholipid scramblase that increases PS externalization and ADAM10-dependent substrate shedding in COS7 cells (Bleibaum et al., 2019). Twenty-four hours posttransfection, increased ANO6 was observed in both MPSF and NPSF as evaluated by immunoblotting (Figure 5a,d). Concomitant with the ANO6 increase, an increase in PS levels in the outer leaflet of the cell membrane was F I G U R E 2 NPSF express ADAM10, which undergoes cell membrane translocation, but is not activated by ionomycin (IM). (a) Immunoblot of ADAM10, where pro-ADAM10 (pro) and mature ADAM10 (m) were observed in both MPSF (M) and NPSF (N). β-Actin was used as loading control. Results are mean ± SEM; n = 3. For statistical analysis, mean values were compared using an unpaired t-test; *p ≤ 0.05. (b) Immunoblot of cell surface and total mature ADAM10 protein fraction in MPSF and NPSF treated for 1 h with IM (0.5 µM). β-Actin was used as loading control in the total protein fraction. Results are mean ± SEM; n = 3. For statistical analysis, mean values were compared using an unpaired t-test; *p ≤ 0. 05. (c) Pseudocoluor; ADAM10 (green), wheat germ agglutinin (magenta), and NucRed™ Live 647 (blue) staining in MPSF and NPSF, with or without a treatment for 1 h with IM (0.5 µM). White arrowheads indicate green puncta of ADAM10 at the cell membrane following IM treatment. Vehicle: DMSO. Scale bar: 10 µm. (d) MPSF and NPSF were transfected with the AP-tagged ADAM10 substrate BTC. BTC shedding was evaluated after cells were treated for 30 min with a general inhibitor of metalloproteases (batimastat, BB, 10 µM), an ADAM10 inhibitor (GI254023X, GI, 1 µM), or an ADAM10/ADAM17 inhibitor (GW280264X, GW, 1 µM), and then cells were stimulated for 1 h with IM (0.5 µM). Vehicle: DMSO. Results are mean ± SEM; n = 3. For statistical analysis, mean values were compared using an ANOVA and Tukey's post hoc test; *p ≤ 0.05. ADAM10, A disintegrin and metalloprotease 10; BTC, betacellulin; MPSF, mouse primary skin fibroblasts; NPSF, NMR primary skin fibroblasts.
URRIOLA-MUÑOZ ET AL. | 767 F I G U R E 3 (See caption on next page) observed in both MPSF and NPSF (Figure 5b,e). Next, MPSF and NPSF were cotransfected with AP-tagged BTC and ANO6-HA (or GFP as control) for 24 h before evaluating ADAM10 shedding activity. As expected, treatment for 1 h with 0.5 µM IM induced an increase in BTC shedding in both control and ANO6-HA MPSF, which was inhibited by batimastat (BB, 10 µM) and GI254023X (GI, 1 µM); IM-induced BTC shedding was higher in cells overexpressing ANO6-HA (Figure 5c). Furthermore, as shown previously (Figure 2d), in NPSF cotransfected with AP-tagged BTC and GFP, IM 0.5 µM did not induce BTC shedding, however, in cells cotransfected with AP-tagged BTC and ANO6-HA, IM induced BTC shedding, which was inhibited by batimastat (BB, 10 µM) and GI254023X (GI, 1 µM) (Figure 5f). Due to NPSF not surviving at 37°C/20% O 2 (Omerbašić et al., 2016), we considered if the difference in culturing conditions could explain the absence of IM-induced ADAM10 shedding activity in NPSF.
Therefore, we performed the same experiments with MPSF cultured in the same conditions as NPSF (seeding, transfection, and experiment). An increase of PS in the outer leaflet of the cell membrane was found in 32°C/3% O 2 MPSF transfected with the ANO6-HA compared with control (Supporting Information: Figure S4A). Moreover, unlike with NPSF, when cultured at 32°C/ 3% O 2 , MPSF cotransfected with AP-tagged BTC and GFP showed increased BTC shedding following IM treatment, which, as with MPSF at 37°C, was greater in cells overexpressing ANO6-HA (Supporting Information: Figure S4B). Therefore, the culturing conditions alone cannot explain the difference in responses to IM between NPSF and MPSF, although we cannot completely rule out any chronic effect of different culturing conditions. To determine if the higher levels of PS in the outer leaflet of the cell membrane found in NMR SV40-Ras skin fibroblasts compared to NPSF might be due to higher expression of the scramblase ANO6, protein levels of ANO6 were evaluated by immunoblot in mouse and NMR primary and SV40-Ras skin fibroblasts, however no difference was found (Supporting Information: Figure S5). These results demonstrate that NMR ADAM10 is a functional metalloprotease and that the lower level of PS in the outer leaflet of the cell membrane, rather than culturing conditions, is likely responsible for the lack of IM-induced ADAM10 shedding activity in NPSF.

| ADAM10 is involved in the migration of NPSF enhanced by the overexpression of ANO6-HA
Migration of tumor cells is necessary for tumor-cell invasion and metastasis. CD44 signaling has been shown to be involved in cell migration in different cells models, including glioma cells (Koochekpour et al., 1995) and several breast cancer cell lines (Zen et al., 2008). In addition, extracellular cleavage of CD44 promotes migration in different models such as, the pancreatic tumor cell line, MIA PaCa-2 (Kajita et al., 2001), human lung adenocarcinoma A549 cells (Kolliopoulos et al., 2021), and certain human breast carcinoma cell lines (Kung et al., 2012). Interestingly, CD44 can induce its own cleavage by ADAM10 through activating the Rac pathway, as demonstrated in migration studies using glioblastoma and lung adenocarcinoma cell lines (Kolliopoulos et al., 2021;Murai et al., 2004). Therefore, it was next evaluated if overexpression of ANO6-HA in NPSF could enhance cell migration. NPSF were seeded F I G U R E 4 NMR SV40-Ras skin fibroblasts have more phosphatidylserine in the outer leaflet of their cell membranes than NPSF. PS in the outer leaflet of the cell membrane was evaluated through the ratio of Annexin V (RLU) and number of cells (RFU) in (a) mouse and (b) NMR, primary and SV40-Ras skin fibroblasts. Results are mean ± SEM; n = 3. For statistical analysis, mean values were compared using an unpaired t-test; *p ≤ 0.05. NMR, naked molerat; NPSF, NMR primary skin fibroblasts; PS, phosphatidylserine; RFU, relative fluorescence units; RLU, relative luminescence unit.
F I G U R E 3 Ionomycin (IM) induces CD44 cleavage and ADAM10 shedding activity in both mouse and NMR SV40-Ras skin fibroblasts. Immunoblot evaluation of full length and cleavage products of CD44 in (a) mouse and (b) NMR SV40-Ras skin fibroblasts. Cells were treated for 30 min with a general inhibitor of metalloproteases (batimastat, BB, 10 µM), an ADAM10 inhibitor (GI254023X, GI, 1 µM), or an ADAM10/ ADAM17 inhibitor (GW280264X, GW, 1 µM), before stimulating cells for with IM (2.5 µM). Full CD44 protein has a molecular weight of 100 kDa, when CD44 is cleaved, two fragments between~15 and~25 kDa are produced. The upper and lower cleavage product is compared with the full protein levels to generate a ratio of CD44 cleavage/total. β-Actin was used as loading control. Vehicle: DMSO. Results are mean ± SEM; n = 3. For statistical analysis, mean values were compared using an ANOVA and Tukey's post hoc test; *p ≤ 0.05. (c) Mouse and (d) NMR SV40-Ras skin fibroblasts were transfected with the AP-tagged ADAM10 substrate BTC. BTC shedding was evaluated after cells were treated for 30 min with a general inhibitor of metalloproteases (batimastat, BB, 10 µM), an ADAM10 inhibitor (GI254023X, GI, 1 µM), or an ADAM10/ADAM17 inhibitor (GW280264X, GW, 1 µM), and then cells were stimulated with IM, 2.5 µM. Vehicle: DMSO. Results are mean ± SEM; n = 3. For statistical analysis, mean values were compared using an ANOVA and Tukey's post hoc test; *p ≤ 0.05. ADAM10, A disintegrin and metalloprotease 10; BTC, betacellulin; NMR, naked mole-rat.

| DISCUSSION
NMRs are cancer resistant, but the mechanisms underpinning this are still not fully understood (Buffenstein et al., 2022;Hadi et al., 2021).

F I G U R E 5
Overexpression of ANO6-HA rescues ionomycin (IM)-induced ADAM10 shedding activity in NMR primary skin fibroblasts. Immunoblot of ANO6 in untransfected and ANO6 hyperactive (ANO6-HA) transfected (a) MPSF and (d) NPSF. β-Actin was used as loading control. PS in the outer leaflet of the cell membrane was evaluated through the ratio between the Annexin V (RLU) and number of cells (RFU) in cells transfected with ANO6-HA or GFP, as a control, in (b) MPSF and (e) NPSF. Results are mean ± SEM; n = 3. For statistical analysis, mean values were compared using an unpaired t-test; *p ≤ 0.05. (c) MPSF and (f) NPSF were cotransfected with the AP-tagged ADAM10 substrate BTC and ANO6-HA or GFP. BTC shedding was evaluated after cells were treated for 30 min with a general inhibitor of metalloproteases (batimastat, BB, 10 µM) or the ADAM10 inhibitor (GI254023X, GI, 1 µM), and then cells were stimulated for 1 h with IM (0.5 µM). Vehicle: DMSO. Results are mean ± SEM; n = 4 for MPSF and n = 3 for NPSF. For statistical analysis, mean values were compared using an ANOVA and Tukey's post hoc test; *p ≤ 0.05. ADAM10, A disintegrin and metalloprotease 10; BTC, betacellulin; MPSF, mouse primary skin fibroblasts; NMR, naked mole-rat; NPSF, NMR primary skin fibroblasts; PS, phosphatidylserine; RLU, relative luminescence unit.
ADAM10 is of particular interest in cancer, being highly expressed, upregulated, and involved in patients with glioblastoma (Siney et al., 2017), lung cancer (J. Guo et al., 2012), and pancreatic cancer (Gaida et al., 2010) to name but a few; ADAM10 overexpression also being associated with a worse prognosis for pancreatic and lung cancers (Trerotola et al., 2021). We therefore evaluated expression and shedding activity of ADAM10 to understand if the absence of IM-induced CD44 cleavage observed in NPSF was simply due to lack of ADAM10 expression. Comparing ADAM10 protein expression in MPSF and NPSF, higher levels of pro-ADAM10, but not mature ADAM10, were found in NPSF. The cleavage site of ADAM substrates is located close to the cell surface (Horiuchi, 2013), and we found that IM induced ADAM10 translocation in both MPSF and NPSF, as well as in the transformed mouse and NMR SV40-Ras skin fibroblasts. Thus, a lack of IM-induced ADAM10-dependent CD44 cleavage is not due to lack of ADAM10 expression or aberrant plasma membrane translocation. Measuring ADAM10-dependent BTC shedding, as another approach to evaluate ADAM10 activity, we found that IM induces BTC shedding in MPSF, mouse SV40-Ras skin fibroblasts, and in NMR SV40-Ras skin fibroblasts, but not in the NPSF, results consistent with the absence of IM-induced CD44 cleavage in NPSF.
Cell membrane lipid composition regulates the shedding activity of ADAM10, such that low cholesterol levels in the cell membrane increase ADAM10 shedding activity, whereas cholesterol enrichment does not (Matthews et al., 2003). Interestingly, the NMR brain has higher cholesterol levels than the mouse brain (Frankel et al., 2020), which may help to explain why under basal conditions there are F I G U R E 6 ADAM10 participates in the enhanced migration of NPSF induced by ANO6-HA overexpression. (a) NPSF were transfected with ANO6-HA or GFP. Twenty-four hours later, the silicone insert was removed and ADAM10 inhibitor, GI254023X (GI, 1 µM), or DMSO (vehicle) was added. Images were taken at 0, 3, 6, 9,12, 24, 36, and 48 h after removal of the silicone insert for gap creation. Scale bar: 200 µm. (b) Quantification of the wound closure over the time is showed. Results are mean ± SEM; n = 3. For statistical analysis, mean values were compared using a two-way ANOVA and Tukey's post hoc test; *p ≤ 0.05 between GFP and ANO6-HA, #p ≤ 0.05 between ANO6-HA and ANO6-HA + GI. ADAM10, A disintegrin and metalloprotease 10; NPSF, NMR primary skin fibroblasts. lower protein levels of CD44 cleavage products and also an absence of IM-induced ADAM10-dependent CD44 cleavage or BTC shedding in NPSF. A further factor regulating ADAM10 shedding activity is PS exposure in the outer leaflet of the cell membrane. The cationic amino acid residues in the stalk region of ADAM10 are thought to interact with the negatively charged PS head group to trigger ADAM10 sheddase activity (Bleibaum et al., 2019). Using mass spectrometry, no difference in the concentration of PS from NMR and mouse total brain lipid extract was detected (Frankel et al., 2020).
However, here we found approximately threefold higher levels of PS exposure on the cell surface of transformed NMR SV40-Ras skin fibroblasts compared to NPSF, but no changes in the PS externalization in mouse SV40-Ras compared with the MPSF. By comparison, PS exposure on the outer leaflet of the cell membrane in NPSF is only about 10% compared to the level in MPSF. This lower level of PS externalization found in NPSF could explain the lower basal protein levels of CD44 cleavage products and the absence of IM-induced CD44 cleavage and ADAM10 shedding activity in NPSF. Consistent with this, when we overexpressed a hyperactive form of ANO6, a scramblase that regulates the PS exposure in the outer leaflet of the cell membrane (Bleibaum et al., 2019), we observed an increase in the PS exposure in NPSF, and as expected, IM-induced ADAM10 shedding activity was "rescued" in these cells as evaluated by the shedding of BTC. Although a higher level of PS exposure at the cell membrane was found in NMR SV40-Ras cells compared to NPSF, no difference was observed in the protein levels of ANO6, one of the scramblases that is responsible of the exposure of PS to the outer leaflet of the cell membrane (Suzuki et al., 2010). However, the abundance of ANO6 is not the only factor to consider and further analysis is required to determine if perhaps processes regulating ANO6 activation are different between cell types. Furthermore, PS exposure is not only dependent on ANO6, other scramblases, such as PLSCR (Zhou et al., 1997) and XKR family members also participating in this process (Suzuki et al., 2013). Moreover, there are also a plethora of flippases and floppases that regulate phospholipid translocation, and thus might also contribute to the raised PS levels observed in NMR SV40-Ras cells compared to NPSF (Daleke, 2003).
It has been shown that CD44 cleavage promotes tumor cell migration (Kawano et al., 2000;Kolliopoulos et al., 2021;Kung et al., 2012;Sugahara et al., 2003) and that ADAM10-dependent CD44 cleavage promotes migration of pituitary adenoma (Pan et al., 2012), glioblastoma (Murai et al., 2004), and melanoma cells (Anderegg et al., 2009). In line with these findings, in a cell migration assay, we observed that the percentage wound closure in NPSF was lower than that of MPSF, but that greater closure occurred following overexpression of ANO6-HA, which was prevented by the ADAM10 inhibitor, GI254023X, thus suggesting that ADAM10 shedding activity might contribute to the NPSF migration.
Taking all these data together, our findings suggest that the lower level of PS in the outer membrane of NPSF prevents ADAM10 shedding activity in response to IM. Future work will be focused on understanding the regulation of PS localization in NPSF, including the roles of scramblases, flippases, and floppases, as well as studying the regulation and localization of Ca 2+ in the NPSF compared to MPSF.
To fully establish whether diminished ADAM10 activity in MPSF contributes to their cancer resistance, in vivo studies would be the next appropriate step.