Off-target effects of protein tyrosine phosphatase inhibitors on oncostatin M-treated human epidermal keratinocytes: the phosphatase targeting STAT1 remains unknown

Materials

Phenyl vinyl sulfonate and phenyl vinyl sulfone (PVS) were purchased from Enamine Ltd (Monmouth, NJ). JTT-551 (PTP1B and TCPTP inhibitor; IC50 = 0.2 and 2 µM, respectively), NSC-87877 (SHP-1 and SHP-2 inhibitor; IC50 = 0.36 and 0.32 µM, respectively), and CX08005 (PTP1B and TCPTP inhibitor; IC50 = 0.78 and 0.48 µM, respectively) were obtained from Millipore Sigma (Darmstadt, Germany). β-actin mouse monoclonal antibody (clone AC-74, catalog number A5316) was obtained from Sigma-Aldrich (St. Louis, MO). STAT1 (rabbit monoclonal, catalog number 9175), phospho-STAT1 (tyrosine 701, rabbit monoclonal, catalog number 9167), phospho-STAT3 (tyrosine 705, rabbit monoclonal, catalog number 9131), Caspase-3 (rabbit monoclonal, catalog number 14220), anti-biotin HRP linked (goat, catalog number 7075), anti-rabbit (polyclonal) and anti-mouse (polyclonal) antibodies and cytokines were purchased from Cell Signaling Technology (Danvers, MA). Sodium vanadate was dissolved in water, sterile filtered and stored at 4 °C until use (Hill III & Rice, 2018).

Cell culture

Spontaneously immortalized human epidermal keratinocytes (SIK) (Rice et al., 1993) were grown in a mixture of Dulbecco’s modified Eagle’s and F12 media (2:1) containing fetal bovine serum (5%), hydrocortisone (0.4 µg/ml), adenine (0.18 mM), transferrin (5 µg/ml) and insulin (5 µg/ml) and co-cultured with a feeder layer of lethally irradiated 3T3 cells (Allen-Hoffmann & Rheinwald, 1984). The medium was supplemented with cholera toxin (10 ng/ml) (EMD Biosciences, La Jolla, CA) at inoculation and with epidermal growth factor (10 ng/ml) at the first and subsequent medium changes. The medium was changed at 3 to 4-day intervals. For inhibitor treatments, cultures (≈90% confluent) were treated with the indicated PTP inhibitor for given times, the medium containing the inhibitor was removed, and OSM was added in prewarmed fresh medium at 50 ng/ml for indicated times.

Differentiation and colony forming efficiency measurements

Newly confluent cultures were treated with 10 ng/ml IL4, IL6, IL20, or 50 ng/ml OSM. These commonly used concentrations were confirmed to give roughly equivalent phosphorylation of STAT1 in the SIK cultures after one hr of treatment. For measurement of differentiation markers, cells were dissolved in Trizol reagent (Invitrogen) after 3–6 days of treatment, and cDNA was synthesized using high-capacity cDNA Reverse Transcription kit (Applied Biosystems). mRNA levels were measured using real-time PCR with TaqMan Fast PCR Master Mix, TaqMan Gene Expression assays (Applied Biosystems) and ABI 7500 Fast Sequence Detection System. Relative mRNA levels were calculated after normalization of each sample to MAPK1 mRNA. Colony forming efficiency was measured after 10 days of treatment. To this end, 3,000 cells were passaged in 6-cm dishes with a lethally irradiated 3T3 feeder layer, grown until colonies were large enough to see easily (7–14 d), fixed and stained with 0.1% rhodamine in water for counting.

Western blotting

Cultures were harvested into a buffer containing 2% w/v SDS, 62.5 mM Tris (pH 6.8), 10% glycerol, with added 5 mM sodium pyrophosphate and 50 µM sodium vanadate to inhibit phosphatase activity. Samples were boiled for 5–7 min and sonicated. Protein was measured with the bicinchoninic acid (Pierce Rockford, IL) (Smith et al., 1985) before incubation with 20 mM dithiothreitol for 20 min at 37 °C. Equal amounts of protein (16–20 µg) were separated by 10% SDS polyacrylamide gel electrophoresis and transferred to Immobilon membranes (Millipore, Danvers, MA). The membranes were blocked with 5% dry milk in Tris-buffered saline and 0.05% Tween 20, followed by incubation with primary antibodies and horseradish peroxidase-conjugated secondary antibodies. Immunopositive bands were detected using ECL Plus Chemiluminescence detection reagent (Pierce, Rockford, IL), detected and quantitated using a Thermo myECL imager (Waltham, MA). Exposure time for each experiment ranged between 1 to 44 min. β-actin served as a loading control.

Assay for transglutaminase activity

Cultures were harvested at confluence into a buffer containing 10 mM Tris (pH 8), 1 mM EDTA and immediately sonicated. Inhibitors and 3 mM CaCl₂ were incubated for 10 min at 37 °C with aliquots of cell sonicate, followed by 2 mM 5-(biotinamido)pentylamine (Lee et al., 1992) for 30 min. Samples were suspended in SDS buffer containing 20 mM dithiothreitol, 20 µg of protein were subjected to SDS polyacrylamide gel electrophoresis and transferred to Immobilon membranes. The membranes were blocked with 5% dry milk in Tris-buffered saline and 0.05% Tween 20, followed by incubation with HRP linked anti-biotin. Immunopositive bands were detected using ECL Plus Chemiluminescence detection reagent and quantitated (Johnson et al., 2012) using a Thermo myECL imager with Thermo My Image Analysis software. Band values were normalized to those from the untreated samples.

Statistical analysis

Statistical significance (p = 0.05) was calculated by one-way ANOVA (Stata/SE9.2 software for Windows) or two-way ANOVA (Wessa, 2020) with Tukey HSD testing.

Striking keratinocyte responses to OSM and representative cytokines

OSM was compared to the cytokines IL4, IL6 and IL20 in effects on cultured human epidermal cells. As shown in Fig. 1A, IL6 and OSM each suppressed the differentiation markers keratin 1, keratin 10 and filaggrin. In contrast to IL4, which was markedly stimulatory toward desmocollin 1, IL6, IL20 and OSM suppressed expression of the junctional proteins desmocollin 1 and desmoglein 1. In each case, OSM produced a striking degree of suppression. When the cultures were treated for 10 days starting at confluence, at which time colony forming ability markedly declines (Patterson et al., 2005), OSM and IL6 partially preserved the cellular colony forming ability (Fig. 1B), evidently slowing the departure of stem-like cells from the germinative pool. The results with IL4 and IL20 also displayed a trend (not statistically significant) toward this effect. OSM was employed for the succeeding experiments.

Vanadate maintenance of STAT1 phosphorylation only at high concentration

Induction of STAT1 phosphorylation on tyrosine 701 by OSM was tested in SIK cultures. As shown in Fig. 2A, the stimulation was transitory, reaching a maximal level within the first hour of OSM treatment and returning close to background level by 6 h. This pronounced decline in phosphorylation was in contrast to the stability of the phosphorylation induced in STAT3, which remained near maximal level for three days (Fig. 2B).

To test the possibility that vanadate could augment cytokine signaling in keratinocytes by preserving STAT1 phosphorylation, SIK cultures were treated for one day in the presence of various concentrations of sodium vanadate and then for one day with OSM. As shown in Fig. 2C, little phosphorylation of STAT1 was detected from treatment for 24 hr with OSM alone. A detectable level of phosphorylation was seen in combination with 30 µM vanadate and a substantially higher level with 100 µM vanadate, while 10 µM vanadate was ineffective. Vanadate alone was also ineffective, and when cultures were treated with 100 µM vanadate for only two hr, followed by a medium change and treatment with OSM for 6 hr, STAT1 phosphorylation was not augmented (Fig. S1).

Selective PTP inhibitors did not prevent STAT1 dephosphorylation

To gain information about the hypothetical PTP activity responsible for reversing the cytokine-induced phosphorylation of STAT1, cultures were treated with OSM alone or in the presence of the inhibitors NSC-87877, a selective inhibitor of SHP2 (Chen et al., 2006), or JTT-551, a known inhibitor of PTP1B (Fukuda et al., 2010). The inhibitors neither affected the level of phospho-STAT1 when administered alone nor gave higher levels of phospho-STAT1 when administered with OSM. Figure 3A shows the lack of effect at concentrations an order of magnitude higher than the IC50s toward PTP1B or SHP-1 and SHP-2. Although TCPTP has been reported to dephosphorylate STAT1 in HeLa cells (ten Hoeve et al., 2002), increasing inhibitor concentrations to 100 µM in SIK did not affect the result (Fig. S2). In addition, when administering CX08005, a more potent competitive inhibitor for PTP1B and TCPTP (Zhang et al., 2016) at various concentrations with OSM, both phospho-STAT1 and phospho-STAT3 were suppressed (and total STAT1 appeared reduced) at the highest concentration (Fig. 3B). The results provide no support for SHP2, TCPTP or PTP1B affecting phospho-STAT1 signaling in SIK cultures by OSM.

Off-target effects of phenyl vinyl sulfonate and phenyl vinyl sulfone

Two members of a more general class of PTP inhibitor were examined for their effect on STAT1 phosphorylation. Aryl vinyl sulfonates and sulfones are directed to the active site sulfhydryls in these phosphatases (Liu et al., 2008). Phenyl vinyl sulfone treatment had little effect, but phenyl vinyl sulfonate (PVSN) induced a striking increase in phospho-STAT1 in the absence of OSM treatment as shown in Fig. 4A. The effect was maximal at 500 µM in the presence or absence of OSM treatment, and considerably lower at 250 µM. By contrast, PVS had little effect on phospho-STAT1 level (Fig. 4B).

In addition to an increase in phospho-STAT1 (90–100 kDa), Fig. 4B showed an even more strongly immunoreactive band at 70–80 kDa detected by the antibody staining. Moreover, in the presence of either PVSN or PVS, the band of phospho-STAT3 seen with OSM treatment alone was eliminated. The appearance of phospho-STAT1 with altered mobility could plausibly result from limited proteolysis by caspase-3 (King & Goodbourn, 1998), an activity that might degrade phospho-STAT3. Cell extracts were therefore examined for activated caspase-3, a potential source of proteolytic activity and an indicator of apoptosis, but only procaspase-3 was detected (Fig. S3).

Prevention of keratinocyte transglutaminase cross-linking

To find whether the cultures survived treatment with PVSN, cells were tested for formation of cross-linked envelopes, a distinctive feature of keratinocyte terminal differentiation that occurs when the plasma membrane becomes permeable at cell death (Green, 1979). At the most effective inhibitor concentration for induction of STAT1 phosphorylation (500 µM), few spontaneous envelopes were observed as a consequence of treatment. To check whether the cells were capable of envelope formation, cultures were treated with the ionophore X537A, known to induce envelope formation in a large majority of keratinocytes in such cultures (Rice & Green, 1979). As shown in Fig. 5A, ionophore-induced envelope formation was inhibited in a concentration-dependent manner. PVS was also effective but less potent, as shown, whereas vanadate (120 µM), NSC-87877 (20 µM) and JTT-551 (20 µM) neither prevented (Fig. S4) nor induced envelope formation under the same conditions.

TGM1, which forms envelopes by cross-linking proteins at the plasma membrane through isopeptide bonds (Rice & Green, 1979), can be assayed by measurement of aliphatic amine incorporation into protein substrates. To confirm TGM1 was inactivated by PVSN and PVS treatment, incorporation of a biotinylated amine transglutaminase substrate was measured in cell extracts. As shown in Fig. 5B, total biotinylated protein was lowered in both PVSN and PVS treated cell extracts at increasing concentration and essentially prevented at 500 µM. While exhibiting similar potencies in cell extracts, the inhibitors could differ in potencies of suppressing envelope formation due to different rates of entry into cells.