The Absence of Tryptase Mcpt6 Causes Elevated Cellular Stress in Response to Modulation of the Histone Acetylation Status in Mast Cells

Mast cells contain large amounts of proteases stored within their secretory granules. Previously we showed that one of these proteases, tryptase, in addition to its location within granules, can also be found within the mast cell nucleus, where it has the capacity to affect the acetylation profile of nucleosomal core histones in aging cells. Based on this notion, and on the known sensitivity of mast cells to modulation of histone acetylation, we here asked whether tryptase could impact on the responses against cellular stress caused by disturbed histone acetylation status. To address this, wild-type and tryptase-deficient (Mcpt6−/−) mast cells were subjected to cell stress caused by trichostatin A (TSA), a histone deacetylase inhibitor. Wild-type and Mcpt6−/− mast cells were equally sensitive to TSA at an early stage of culture (~8 weeks). However, in aging mast cells (>50 weeks), tryptase-deficiency led to increased sensitivity to cell death. To address the underlying mechanism, we assessed effects of tryptase deficiency on the expression of markers for proliferation and cell stress. These analyses revealed aberrant regulation of thioredoxin, thioredoxin reductase, glutaredoxin, and glutathione reductase, as well as blunted upregulation of ribonucleotide reductase subunit R2 in response to TSA in aging cells. Moreover, the absence of tryptase led to increased expression of Psme4/PA200, a proteasome variant involved in the processing of acetylated core histones. Altogether, this study identifies a novel role for tryptase in regulating the manifestations of cell stress in aging mast cells.

A hallmark feature of MCs is their remarkably high content of secretory granules. These are filled with a plethora of preformed inflammatory mediators, including histamine and other bioactive amines, cytokines, growth factors, lysosomal hydrolases serglycin proteoglycans, and a panel of MC-restricted proteases. The latter include chymases, tryptases, and carboxypeptidase A3 (CPA3) [17], of which tryptases and chymases are serine endo-proteases whereas CPA3 is a metallo-exopeptidase [18][19][20].

May-Grünwald/Giemsa Staining
To prepare cytospin slides, 100 µL of cell suspensions were centrifuged onto the slides for 5 min at 500 rpm. The slides were air-dried and incubated with 100% May-Grünwald Eosine-methylene blue solution for 5 min and then with 50% May-Grünwald Eosine-methylene blue solution for 1 min, followed by 15 min incubation in 2.5% Giemsa Azur Eosin-methylene solution and washing in H 2 O. The slides were dried before mounting. Experiments were repeated with three different batches of cells.

Cell Viability
Cells were washed and resuspended in Annexin V binding buffer (BD Biosciences, Franklin Lakes, NJ, USA) and stained with Annexin V (BD Biosciences) and DRAQ7™ (Biostatus Ltd., Shepshed, UK). Subsequently, stained cells were analyzed with an Accuri flow cytometer (BD Biosciences) for assessment of cell death. Data analysis was performed using the FlowJo software (TreeStar Inc., Ashland, OR, USA).

Quantitative Real-Time PCR
NucleoSpin ® RNA isolation kit from (MACHEREY-NAGEL GmbH & Co. KG) was used for total RNA isolation. RNA purity and concentrations were assessed with a Nanodrop device. Amounts of 200 ng of highly purified RNA (A260/280 > 1.95) were used for cDNA synthesis (Bio-Rad, Solna, Sweden). SYBR GreenER SuperMix was used along with primer mix and cDNA for the quantitative real-time PCR

Confocal Microscopy
Cytospin slides were prepared from aliquots (100 µL) of cell suspensions and cells were then fixed with 4% paraformaldehyde in PBS for 15 min. An amount of 100 µL of 50 µg/mL digitonin solution in PBS was added to each glass slide and incubated for 10 min at room temperature. Next, 100 µL anti-Psme4/PA200 antibody (1:500) in TBS/1% BSA and/or isotype control at the same concentration were added and left overnight at 4 • C, followed by washing 3 times with TBS-T. Next, Alexa-conjugated secondary antibody diluted (1:1000) in TBS/1% BSA was added and incubated for 1 h at room temperature. ActinRed/Green TM (Molecular Probes) probes were used to stain cells. The slides were kept in the dark, and washed 3 times with TBS-T between each step. Finally, 100 µL of Nucblue TM (Molecular Probes) probes in TBS/1% BSA was added for 20 min, followed by 3 times washing with TBS-T. The slides were mounted with SlowFade ® gold antifade mounting medium (Life Technologies). Samples were analyzed using a laser-scanning microscope equipped with ZEN 2009 software (LSM 710; Carl Zeiss, Berlin, Germany).

Flow Cytometry
MCs (0.5 × 10 6 cells/mL) were fixed and permeabilized using eBioscience TM intracellular fixation and permeabilization buffer set (eBioscience, San Diego, CA, USA). Subsequently, cells were incubated with rabbit anti-Psme4/PA200 antibody (1:250) and/or isotype control at the same concentration for 1h at room temperature. Cells were then washed and incubated with anti-rabbit Alexa488 antibody (1:1000) for 30 min at room temperature. After washing twice, cells were resuspended to 500 µL in PBS and assessed by flow cytometry (BD Accuri C6 plus, BD bioscience, San Jose, CA, USA). Data analysis was performed using the FlowJo software (TreeStar Inc., Ashland, OR, USA).

Results
To study the impact of HDAC inhibition on MCs, and if such effects are dependent on tryptase, we developed bone marrow-derived MCs from wild-type (WT) and tryptase-deficient (Mcpt6 −/− ) mice. Cells were cultured for extended time periods, to allow an assessment of age-dependent effects of HDAC inhibition on MCs. Morphological assessments showed that both the WT and Mcpt6 −/− MCs were, as expected, densely granulated ( Figure 1A). Moreover, the extent of granulation was similar in the early stage and aged cells ( Figure 1A). However, it was noted that Mcpt6 −/− cells were somewhat larger than corresponding WT cells ( Figure 1A). Importantly, almost 100% of the cells, both from WT and Mcpt6 −/− mice were positive for the high affinity IgE receptor (FcεRI), confirming that they represent differentiated MCs ( Figure 1B,C). In early stage cultures (~8 weeks), both WT and Mcpt6 −/− MCs were highly sensitive to long-term treatment with HDAC inhibitor (Trichostatin A; TSA). As seen in Figure 2A, TSA caused a reduction in the number of viable cells, with approximately equal effects seen in cultures of WT and Mcpt6 −/− cells. In agreement with this, TSA caused an increase in the proportion of dead cells as measured by Annexin V positivity (AnnV + ), with similar effects seen on WT and Mcpt6 −/− cells ( Figure 2A). In contrast, the effects of TSA were different on the aged (~50 weeks) WT vs. Mcpt6 −/− cells. As seen in Figure 2B, TSA caused a significant increase in the number of viable aged WT cells, whereas a significant reduction of the number of viable cells was seen in aged Mcpt6 −/− cells. These effects were reflected by corresponding changes in cell populations showing Annexin V positivity, i.e., TSA caused a decrease in the Annexin V positivity of aged WT cells, but caused an increase in Annexin V positivity in aged Mcpt6 −/− cells ( Figure 2B). Hence, the absence of tryptase (Mcpt6) results in an increase in the sensitivity to HDAC inhibition, and this occurs in an age-dependent manner. This introduced the possibility that tryptase can have an impact on responses to cell stress caused by HDAC inhibition. To provide further insight into this we assessed whether tryptase could regulate the expression of genes operative in regulating oxidative stress. To this end we analyzed the effect of TSA on the expression of thioredoxin 1 (Trx1), thioredoxin reductase (TR), glutaredoxin 1 (Grx1), and glutathione reductase (GR) in WT vs. Mcpt6 −/− MCs. Trx1 is a redox active protein involved in defense against oxidative damage and TR is an enzyme having the function to reduce Trx1, thereby being essential for TR function. Grx1 and GR are both part of the glutathione antioxidative system [31,32]. When subjecting the early stage MCs to TSA, it was seen that WT cells were refractory in terms of Trx1 upregulation. In contrast, Mctp6 −/− cells responded to TSA with a robust upregulation of Trx1 ( Figure 3A). Both WT and Mcpt6 −/− cells showed significant upregulation of TR expression in response to TSA ( Figure 3B). In the aged cells, no significant effects of TSA on Trx1 or TR expression was seen in Mcpt6 −/− cells, whereas both Trx1 and TR were significantly downregulated in TSA-treated WT cells ( Figure 3A,B). When examining the effects of TSA on Grx1 and GR, similar patterns as those seen for Trx1/TR were observed, i.e., aged WT cells responded to TSA by down-regulating these genes, whereas Mcpt6 −/− cells were either refractory (GR) or showed a slightly increased expression (Grx1). At the early stage, both WT and Mcpt6 −/− cells responded to TSA by up-regulating both genes ( Figure 4A,B). Hence, these findings indicate that the absence of tryptase affects the ability of MCs to respond to cellular stress through induction of anti-oxidative mechanisms.
The findings above suggested that tryptase regulates cell numbers in response to TSA, which could potentially be linked to effects on proliferation. To address this, we investigated if tryptase can influence the expression of ribonucleotide reductase (RNR), a key enzyme in the generation of deoxyribonucleotides necessary for DNA synthesis. Notably, RNR is dependent on the Trx1/TR and Grx1/GR systems for activity, and the differential regulation of these enzymes in WT and Mcpt6 −/− MCs treated with TSA could thus reflect differential RNR function. RNR is composed of two subunits, R1 and R2. In the cell cycle S phase, canonical R2 is predominantly expressed but is replaced by p53R2 in quiescent cells (G 0 phase). A high R2/p53R2 ratio is thus significative for proliferating cells, typically seen in transformed cells [31].  After exposure to TSA, early stage WT MCs responded by upregulation of all of these RNR subunits ( Figure 5A-C). In the Mcpt6 −/− early stage cells, R1, R2, and p53R2 were also significantly induced by TSA treatment. However, it was noted that the TSA-induced upregulation of R1 and p53R2 was weaker than in WT cells, whereas the Mcpt6 −/− cells responded more vividly than WT cells in terms of R2 induction ( Figure 5A-C).
In aged MCs, TSA caused a significant increase in R1 expression in Mcpt6 −/− cells but, in contrast, caused a reduction of the R1 expression in WT cells ( Figure 5A). Similarly, p53R2 expression was reduced in TSA-treated WT cells but was significantly increased in corresponding Mcpt6 −/− MCs ( Figure 5C). In contrast, R2 expression was stimulated in aged WT cells but was decreased to a major extent in cells lacking tryptase ( Figure 5B). Hence, these findings suggest that tryptase has an impact on the regulation of the different RNR subunits in response to cellular stress caused by HDAC inhibition. Altogether, these data suggest that tryptase has a major impact on the cellular responses caused by disturbed acetylation status of nuclear core histones. Thereby, we hypothesized that tryptase could play a role in regulation of mechanisms operative in maintaining a proper balance of acetylated histones. To date, there is very little insight into how this balance can be regulated. However, it has been shown that the PA200 proteasome (encoded by Psme4) can fulfill such a function in sperms [33], and also can have a similar role in transformed cell lines [34]. However, it is not known whether Psme4/PA200 is expressed in primary immune cells such as MCs and whether Psme4/PA200 can be linked to regulation of the histone acetylation in such cells. To approach this, we analyzed for the expression of Psme4/PA200 in MCs and asked whether Psme4/PA200 expression is affected by TSA and is influenced by the absence/presence of tryptase. As seen in Figure 6A, baseline Psme4/PA200 mRNA expression was seen in both the early stage and aged MCs. After treatment with TSA it was observed that Mcpt6 −/− cells responded by upregulation of Psme4/PA200 mRNA expression, whereas WT cells were either refractory (early stage cells) or showed downregulated expression (aged cells) ( Figure 6A). To provide support for these findings at the protein level we first performed flow cytometry analyses. These analyses confirmed that early stage WT MCs did not significantly upregulate the expression of Psme4/PA200 at the protein level in response to TSA, whereas a trend of upregulated Psme4/PA200 was seen in corresponding Mcpt6 −/− cells ( Figure 6B,C). In agreement with the mRNA analysis, aged WT cells showed downregulated expression of Psme4/PA200 protein in response to TSA whereas, in contrast, TSA caused an upregulation of Psme4/PA200 protein in Mcpt6 −/− cells ( Figure 6B,C). We also used confocal microscopy to visualize effects of TSA on Psme4/PA200. As shown in Figure 7, relatively weak baseline staining for Psme4/PA200 was seen in both WT and Mcpt6 −/− early stage MCs. After treatment with TSA, Psme4/PA200 staining was somewhat increased. In aged WT cells, TSA caused a profound decrease in Psme4/PA200 staining (Figure 7), in agreement with the qPCR and flow cytometry data. In contrast, TSA caused an increase in Psme4/PA200 staining in aged Mcpt6 −/− cells (Figure 7), which is in agreement with the qPCR and flow cytometry data. Hence, the absence of Mcpt6 is associated with aberrant expression of Psme4/PA200 proteasomes in response to cell stress caused by interference with histone deacetylation. that were either cultured alone (Control) or with TSA as indicated, followed by staining with DAPI (blue), Psme4/PA200 (green) and actin (red). The lower panels represent staining with the isotype control for the Psme4/PA200 antibody.

Discussion
MC tryptase has unique macromolecular properties, being built up as a tetramer where all of the active sites are facing a narrow central pore. Thereby, tryptase resembles the organization of proteasomes [35,36]. Due to this organization, tryptase is unique among serine proteases by being resistant to endogenous protease inhibitors. Tryptase activity is thus refractory to milieus rich in protease inhibitors such as plasma and cytosolic compartments. The regulation of tryptase activity is instead thought to occur through spontaneous, time-dependent dissociation of the tryptase tetramer, which leads to enzymatic destabilization [37,38]. Tryptase has previously been linked to the pathology of asthma (reviewed in [10]) and there are also reports implicating tryptase in a range of other settings, including tumor angiogenesis [39], melanoma cell proliferation [40], chronic obstructive pulmonary disease [41], arthritis [42], and acute experimental colitis [43].
The dogma has for a long time been that tryptase is exclusively localized to the MC secretory granules, and that the biological functions of tryptase are dependent on its release from MCs followed by effects on other cell types or on compounds residing in extracellular environment [17,19,38]. However, we recently challenged this notion by showing that tryptase, in addition to its location within the MC secretory granules, also can be found within the MC nucleus [25,28]. Moreover, we showed that tryptase can have an extensive functional impact on the MC itself, i.e., that tryptase can have cell-intrinsic functions in addition to its canonical cell-extrinsic functions [25,28]. When deciphering such cell-intrinsic functions, we noted that tryptase can regulate the proliferation of MCs [28], but the underlying mechanism was not revealed at that time. Moreover, we made the intriguing observation that tryptase has the ability to process the N-terminal ends of nuclear core histones, thereby removing epigenetic acetylation marks [25,28]. Another intriguing observation was that these effects were seen in aging MCs, but not in early stage MC cultures.
Based on these earlier observations, the present investigation sought to provide further insight into the cell-intrinsic properties of tryptase, with a particular focus on how tryptase affects processes related to regulation of histone acetylation, and if such effects are cell age-dependent. In support of such a hypothesis, previous studies have indicated that MCs are remarkably sensitive to HDAC inhibition [29], suggesting that an extensive accumulation of acetylated histones causes cellular stress to MCs.
Since our previous studies indicated that tryptase has the ability to regulate MC function in a manner dependent on histone acetylation [28], we reasoned that tryptase might have an impact on responses downstream of HDAC inhibition. Indeed, we show here that the absence of tryptase leads to increased sensitivity of MCs to toxicity induced by HDAC inhibition. Further, in agreement with our earlier observation that cell-intrinsic effects of tryptase are age-dependent, increased sensitivity of tryptase-deficient MCs to HDAC inhibition was only seen in MCs that had undergone extensive aging. The mechanism behind these findings is intriguing. A likely scenario, based on our previous work, is that the elevated sensitivity of Mcpt6 −/− MCs to HDAC inhibition might be due to differential levels of epigenetic histone acetylation marks in WT vs. Mcpt6 −/− MCs. Hence, this introduces the possibility that MC homeostasis in an aging context is dependent on adequate regulation of histone acetylation. This could be potentially be accomplished by regulation of enzymes that catalyze the acetylation/deacetylation of core histones. Alternatively, regulation of histone acetylation could be executed by proteolytic processing of core histones, whereby epigenetic histone marks are erased. The latter possibility has received very little attention, but there is emerging evidence from other cell types (sperms, transformed cell lines) that proteasomal degradation of core histones could serve such a function [33,34]. In this respect, proteasomes of the Psme4/PA200 type were recently shown to degrade nuclear histones and it was proposed that such degradation could serve to regulate the epigenetic profile of the cell [34]. Based on those observations we here hypothesized that the absence of tryptase could affect the levels of Psme4/PA200 proteasomes, thereby disturbing the appropriate turnover of core histones. In agreement with this notion, we show that the absence of tryptase leads to aberrant expression of Psme4/PA200. In agreement with effects of tryptase being cell-age dependent, aberrant expression of Psme4/PA200 was predominantly seen in aging MCs.
Based on these findings, we may propose that tryptase has an important role in maintaining an adequate status of acetylated histones, and thereby that toxic responses to HDAC inhibition are relatively modest in tryptase-expressing WT cells. In the absence of tryptase, the response to HDAC inhibition may thus become exaggerated and the cells might attempt to compensate this unbalance by increasing the expression of histone-degrading Psme4/PA200 proteasome units.
Overall, these findings suggest that tryptase-deficient MCs are more sensitive to cellular stress than are WT cells. As a further sign of this, we show that early stage tryptase-deficient MCs respond to HDAC inhibition by elevated upregulation of Trx1, an enzyme with a prominent role in the defense against oxidative stress. In addition, we noted that Trx1 expression in aged WT MCs was suppressed by HDAC inhibition, but was unaffected in tryptase-deficient cells. Similar patterns were seen also for TR, Grx1, and GR in aged cells, i.e., HDAC inhibition caused downregulation of the respective genes in WT cells whereas tryptase-deficient cells were largely refractory.
In addition to their roles in defense against oxidative stress, both Trx1/TR and Grx/GR are electron donors for RNR, an enzyme that has a key role in cellular proliferation by synthesis of deoxyribonucleotides to be incorporated into DNA. The absence of tryptase could thus, additionally, have an impact on RNR. In line with this, our data show that tryptase-deficiency is associated with differential regulation of the R2 subunit of RNR in response to cellular stress caused by HDAC inhibition. Since R2 (rather than p53R2) expression is typically associated with proliferative responses, it is conceivable that tryptase-dependent effects on R2 expression may account for our previous observation of elevated proliferative activity in MCs lacking tryptase [28].
It is important to emphasize that the findings presented here emanate from MCs that have aged in cell culture, and we are at present not able to ascertain that the observed effects of tryptase-deficiency are replicated in vivo in aging MC populations. However, we have previously shown that the absence of Mcpt6 causes an age-dependent expansion of MC populations in vivo, as well as increased size of MCs [28]. Hence, these findings suggest that tryptase indeed can have an impact on selected features of aging MCs in vivo, although further investigations will be required to specifically assess whether tryptase has an age-dependent impact on sensitivity of MCs to cellular stress in vivo.
In a broader perspective, we may also speculate that mechanisms such as those identified in this work may have an impact on aging in general. Specifically, we may speculate that maintaining of a proper balance of the histone acetylation status is a significant factor in the maintenance of cellular homeostasis, and that aging is associated with a significant challenge for the respective cell to prevent accumulation of epigenetic histone marks that otherwise might cause cellular damage. Possibly, tryptase may serve such a function in a MC context and it is also possible that similar mechanisms are operative in other cell types, although executed by enzymes other than tryptase. Accordingly, if this controlling mechanism is absent, as in tryptase-deficient MCs, aging cells may become more vulnerable to cellular stress as shown here for MCs. However, the validity of this hypothesis on a more general level remains to be investigated.