PRSS3/mesotrypsin as a putative regulator of the biophysical characteristics of epidermal keratinocytes in superficial layers

Mesotrypsin, encoded by the PRSS3 gene, is a distinctive trypsin isoform renowned for its exceptional resistance to traditional trypsin inhibitors and unique substrate specificity. Within the skin epidermis, this protein primarily expresses in the upper layers of the stratified epidermis and plays a crucial role in processing pro-filaggrin (Pro-FLG). Although prior studies have partially elucidated its functions using primary cultured keratinocytes, challenges persist due to these cells' differentiation-activated cell death program. In the present study, HaCaT keratinocytes, characterized by minimal endogenous mesotrypsin expression and sustained proliferation in differentiated states, were utilized to further scrutinize the function of mesotrypsin. Despite the ready degradation of the intact form of active mesotrypsin in these cells, fusion with Venus, flanked by a peptide linker, enables evasion from the protein elimination machinery, thus facilitating activation of the Pro-FLG processing system. Inducing Venus-mesotrypsin expression in the cells resulted in a flattened phenotype and reduced proliferative capacity. Moreover, these cells displayed altered F-actin assembly, enhanced E-cadherin adhesive activity, and facilitated tight junction formation without overtly influencing epidermal differentiation. These findings underscore mesotrypsin's potentially pivotal role in shaping the characteristic cellular morphology of upper epidermal layers.


Generation of HaCaT cell derivatives for functional analyses of mesotrypsin
Expanding on the expression patterns of mesotrypsin and its representative substrate, Pro-FLG, we sought to elucidate the functional role of mesotrypsin in HaCaT cells.Initially, we engineered HaCaT cells harboring a tet/dox-inducible expression construct featuring active mesotrypsin tagged with a T7 peptide at the C-terminus.To achieve this, we employed the tetracycline-responsive element (TRE)-containing plasmid PBtet0606, housing the expression cassette for a neomycin resistance gene independent of tet/dox induction.Additionally, we utilized the reverse tet/dox transactivator (rtTA) plasmid containing a hygromycin resistance gene, along with a piggyBac (PB) transposase plasmid (Fig. 2A).While cells resistant to both antibiotics expressed mesotrypsin mRNA in response to dox treatment, the protein expression of mesotrypsin was detected only in Cos7 cells and not in HaCaT cells (Fig. 2A).This observation suggested that HaCaT cells exhibit either translational inhibition or a protein-eliminating system for active mesotrypsin.Subsequently, we evaluated the expression of the Venus-mesotrypsin fusion protein.The approximately 60 kDa protein was identified using an anti-GFP/Venus antibody in both Cos7 and HaCaT cells, indicating successful expression of the fusion protein.However, a distinct protein band at approximately 38 kDa was detected with the antibody against the N-terminal Venus but not with the C-terminal T7-tag.Additionally, T7-tagged proteins, except for the full-length ~ 60 kDa protein, were not discernible (Fig. 2B).This finding suggested that a significant portion of the mesotrypsin domain in the fusion protein undergoes rapid degradation in HaCaT cells.Treatment with the potent proteasome inhibitor MG132, as opposed to the control DMSO, impeded the degradation of mesotrypsin (Fig. 2C).This finding suggested that HaCaT cells possess posttranslational regulatory systems for the mesotrypsin protein, complementing its transcriptional suppression (Fig. 1B).We then investigated whether the Venus-mesotrypsin fusion protein retains its proteolytic activity.The Pro-FLG protein, a substrate of mesotrypsin processing, exhibited a reduction in abundance and a slight alteration in its molecular weight upon Venus-mesotrypsin expression (Fig. 3A).In addition, the recombinant form of the GFP-mesotrypsin fusion protein, but not GFP alone, degraded the Pro-FLG protein extracted from HaCaT cells, leaving a 76 kDa FLG dimer (Fig. 3B), indicating that the GFP/Venus-mesotrypsin fusion protein, which evades proteasomal degradation, partially exerts effects of mesotrypsin.Notably, however, while mesotrypsin is recognized for cleaving off the N-terminal domain from Pro-FLG exclusively, the amount  of Pro-FLG fragments was dramatically decreased.This suggests that HaCaT cells harbor enzymes that facilitate further processing of these fragments.Indeed, we observed the expression of KLK5, a protease activated by mesotrypsin to further process Pro-FLG fragments into FLG, along with another FLG-processing protease, calpain-1 (Supplementary Fig. 1).Furthermore, it has been reported that FLG is very unstable in HaCaT cells, which express caspase14 to digest FLG into small peptides known as natural moisturizing factors (NMFs) 12 .

Effect of mesotrypsin on HaCaT cell behaviors
Upon induction of Venus-mesotrypsin expression, HaCaT cells exhibited an increase in size, and z-stack image analysis revealed a pronounced manifestation of a flattened morphology, a phenomenon conspicuously absent in Cos7 cells (Fig. 4A,B and Supplementary Fig. S2).Additionally, the Alamar Blue assay illustrated that mesotrypsin induces a significant reduction in the proliferation of HaCaT cells (Fig. 4C).These morphological shifts, coupled with the growth arrest, epitomize hallmark features akin to keratinocytes residing in the granular and cornified layers, practically mirroring the characteristics of fully differentiated NHEK cells 32 .This finding suggested that mesotrypsin plays a pivotal role as a regulator of the morphological attributes distinctive to the differentiated upper layer of the epidermis.

Effect of mesotrypsin on the formation of AJs and TJs
Another distinctive feature of the granular layer is the formation of tight junctions (TJs), which are discernible intercellular adhesion complexes governing paracellular permeability and conferring barrier functions upon the epidermis 33,34 .Considering the prerequisite for stable adherens junctions (AJs) in TJ formation 35 , our initial investigation focused on the impact of mesotrypsin on the physical characteristics of AJs.Our findings indicate that Venus-mesotrypsin elevates the expression of E-cadherin, the primary constituent of epidermal AJs (Fig. 5A).
To evaluate the strength of cadherin-mediated cell-cell adhesion, cells depleted of most cell surface proteins, except for cadherins (Supplementary Fig. S3), underwent mechanical dissociation and subsequent re-aggregation.These cells demonstrated heightened adhesive capabilities following the expression of Venus-mesotrypsin but not Venus alone (Fig. 5B), suggesting that mesotrypsin stabilizes or fortifies adherens junctions in HaCaT cells.Subsequently, examinations were conducted to assess the influence of mesotrypsin on TJ formation.The cytoplasmic translocation of ZO-1, the major scaffolding protein of TJs, is considered a representative hallmark of TJ formation in epidermal keratinocytes and is induced in HaCaT cells by simultaneous treatment with 10 mM calcium and a JNK inhibitor 35,36 .Our findings demonstrated that rapid ZO-1 translocation, along with E-cadherin accumulation at cell-cell contact sites, was induced in HaCaT cells expressing Venus-mesotrypsin even in the absence of elevated calcium concentrations, highlighting the facilitative role of mesotrypsin in TJ formation (Fig. 6A,B).Concurrent with the changes in the localization of critical components of the cell-cell adhesion apparatus, mesotrypsin significantly influenced the dynamics of F-actin, directing stress fiber assembly in HaCaT cells (Fig. 6C).

Effect of mesotrypsin on growth and differentiation
Given that mitotic keratinocytes are not detected within the upper-layer epidermis, we tested the growth profile of the mesotrypsin-expressing HaCaT cells.As mesotrypsin expression induced significant suppression of cell growth (Fig. 4C), we assessed the expression profiles of proteins regulating cell cycle progression, namely, Rb, c-Myc, and P21 37,38 .Rb governs the expression of genes essential for the transition from the G1 to S phase of the  cell cycle, c-Myc orchestrates the transition from G0/G1 to S phase by activating the cyclin-dependent kinase (CDK) complex, and p21 serves as a cyclin-dependent kinase inhibitor that impedes positive feedback loops for G1 progression.Our findings revealed that cells expressing mesotrypsin exhibited a significant reduction in the level of inactive phosphorylated Rb, downregulation of c-Myc, and upregulation of P21, indicating growth arrest in the G1 phase (Fig. 7A).In addition to these effects, the number of Ki67-positive cells was significantly decreased (Fig. 7B), suggesting that mesotrypsin induced cell cycle exit.We detected downregulation of the senescence biomarker lamin B1 while leaving other cognate lamins, such as lamin A/C, unaffected (Fig. 7B).A decrease in the expression of laminB1 is also observed in the upper layers of the skin epidermis 5 .The induction of G1 arrest and cell cycle exit account for the observed effects on cell proliferation.In terms of differentiation, although HaCaT cells maintained for a long period in conventional high-calcium medium retain their differentiation potential 39,40 and mesotrypsin diminishes the abundance of the differentiation marker Pro-FLG (Fig. 3), the expression of canonical differentiation markers, including TGase1, Loricrin, and Involucrin, remains unaffected (Fig. 7C).These findings suggest that mesotrypsin may play a pivotal role in inducing cell cycle exit and shaping the morphological characteristics of upper-layered keratinocytes without significantly impacting the differentiation process.

Discussion
Epidermal keratinocytes exhibit distinct characteristics, specifically undergoing substantial alterations in cell morphology, physical attributes, membrane composition, and intercellular junctional complexes during the differentiation/stratification process 41,42 .To cause such profound changes, spatiotemporal regulation of the functional expression of numerous molecular components is imperative [43][44][45] .Notably, our focus centers on mesotrypsin, given its role as a serine protease involved in processing Pro-FLG to generate FLG-N, a process pivotal for epidermal apoptosis.Additionally, mesotrypsin activates KLKs to produce multiple FLG units, crucial for both aggregating keratin filaments and contributing to the formation of the cornified cell membrane in upper-layer keratinocytes.Moreover, most mesotrypsin isoforms found in keratinocytes lack the signal peptide essential for secretion 23 .Nonetheless, it is conceivable that this subset may be secreted via a non-classical secretion pathway, functioning as an extracellular protease.Indeed, differentiated keratinocytes produce various essential elements that regulate cellular behaviors in both the cytoplasmic and extracellular environments 13,30,31 .
We scrutinized the behaviors of HaCaT keratinocytes, as these cells, in contrast to NHEK cells, express a considerable amount of Pro-FLG with minimal mesotrypsin in conventional culture media.Although exogenous active mesotrypsin seems to undergo rapid degradation in HaCaT cells, prior research has documented the successful production of mesotrypsin in NHEK cells 14,25 .This observation suggested that HaCaT cells may have lost crucial elements for Pro-FLG processing, and this study identifies mesotrypsin as a strong candidate.
In the upper-layer epidermis, active mesotrypsin is derived from its precursor mesotrypsinogen through processing by several proteases, including Cathepsin B, Caspase14, and Enterokinase [23][24][25] .Alternatively, we explored an inducible expression system for the active form of mesotrypsin, given the difficulty in precisely controlling its functional expression in HaCaT cells.While the intact form of exogenous active mesotrypsin is swiftly degraded by the proteasome, fusion with the Venus protein significantly impedes protein elimination.Although mesotrypsin can undergo chymotrypsin C-triggered self-degradation, as observed in other tissues 46 , a subpopulation of mesotrypsin in the fusion protein evades the protein elimination system, appearing to induce several characteristics in HaCaT cells mirroring epidermal upper-layer keratinocytes.
Despite mesotrypsin inducing a flattened morphology in HaCaT cells, no morphological changes were observed in Cos7 cells.These findings suggested that the mesotrypsin targets responsible for this phenomenon are epidermal cell-specific proteins.The cuboidal-squamous transition in epithelial cells is known to be caused by mechanical properties 47,48 , and keratins act as the primary regulators in keratinocytes 7,49 .Given that FLG functions by bundling keratin and promoting epidermal flattening 2,16 , it is conceivable that this effect was induced, at least in part, through the temporal regulation of keratin dynamics caused by the Pro-FLG processing.While a preceding inquiry observed the existence of multinucleated cells in giant HaCaT cells 26 , mesotrypsin did not induce such cellular phenomena.However, notably, the expression level of mesotrypsin and phenotypic manifestation did not entirely correlate (data not shown), likely due to the ongoing proteasomal degradation of exogenous mesotrypsin.
In addition to exhibiting a flattened morphology, keratinocytes in the granular layer distinctively form TJs at the cell-cell boundary, supported by stable AJs 50 .The expression of mesotrypsin upregulated the major AJ compartment E-cadherin to enhance cadherin-mediated intercellular adhesive strength, followed by accelerated membrane translocation of the TJ scaffold protein ZO-1.While the regulation of the expression profile of these intercellular junctional components is influenced by cytoskeletal rearrangement involving F-actin 51 and keratin intermediate filaments 52 , both of which are impacted by mesotrypsin, further experiments are requisite to elucidate the molecular mechanism facilitating tight junction formation under the influence of mesotrypsin.
Another noteworthy effect of mesotrypsin is its ability to induce G1 growth arrest in HaCaT cells.In the epidermis of the skin, mitotic cells are predominantly confined to basal layer keratinocytes, and nascent daughter cells promptly cease the cell cycle upon outward extrusion 53 , where mesotrypsin expression has not yet manifested.This observation implies that the mesotrypsin-induced growth arrest in HaCaT cells does not correspond to the cell cycle exit in the normal epidermis, highlighting the necessity for meticulous investigation to delineate the precise role of mesotrypsin on the keratinocyte proliferation.Nonetheless, considering the presence of proliferative keratinocytes often observed in the upper epidermal layers of precancerous lesions, such as actinic keratosis 54 , the effects elucidated in this study could support the proposition that mesotrypsin possesses an intrinsic capacity to act as a backup regulator for preventing re-entry into the growth cell cycle, even within normal keratinocytes.On the other hand, HaCaT cells expressing mesotrypsin undergo the initiation of cell  www.nature.com/scientificreports/senescence, as evidenced by the significant reduction in laminB1 expression, a phenomenon also observable in the upper layers of the epidermis even in young skin 5 .Keratinocytes exhibit a distinctive quality wherein the same cellular entities undergo significant alterations in their growth profile, cellular morphology, and intercellular junctions during stratification.Despite the prevailing notion in most prior investigations that these physical properties are intricately linked to keratinocyte differentiation, our study illustrates the induction of cells that exhibit physical characteristics associated with upper-layer keratinocytes but are not undergoing differentiation.This finding suggested the potential omission of intricate epidermal differentiation in the examination of keratinocyte cell behaviors, particularly concerning our longterm-maintained HaCaT cells in the conventional high-calcium medium.Nevertheless, this cellular system may afford notable advantages in elucidating the crucial regulators of keratinocyte morphological behaviors, which are synergistically facilitated by easily manipulable gene transfection.

Generation of HaCaT cells with active mesotrypsin
To construct the tetracycline/doxycycline-inducible expression vector for mesotrypsin, cDNA encoding processed active mesotrypsin (from Ile-81 to Ser-304, accession number; P35030) was amplified by PCR using a plasmid (RIKEN, IRAK0 62C01) as the template.Subsequently, the cDNA encoding T7-tag (MYSMQLAS-CVTLTLVLLVN) was fused at the C-terminal sequence, after which the resulting cDNA was inserted into the Not I site in PBtet0606 vector 11,35 to generate PBtet0606-mesotrypsin.For construction of the inducible expression vector for the Venus-mesotrypsin fusion protein, cDNA encoding the entire Venus protein (excluding the stop codon) was sequentially fused with synthetic double-strand DNA encoding a linker sequence (GGGGSGGSGGGSTQGEL), followed by the TEV protease recognition sequence (ENLYFQG) and the cDNA of the processed active mesotrypsin (from Ile-81 to Ser-304).Venus, a modified form of GFP (accession number; P42212), was utilized for facile detection of the transgene in living cells.The resulting DNA construct was subsequently inserted into the PBtet0606 vector, yielding PBtet0606-Venus-mesotrypsin.As a control, the plasmid PBtet0606-Venus lacking the mesotrypsin cDNA sequence from PBtet0606-Venus-mesotrypsin was also prepared.To establish HaCaT cells with dox-inducible expression of mesotrypsin or Venus-mesotrypsin, cells were transfected with PBtet0606-mesotrypsin or PBtet0606-Venus-mesotrypsin in conjunction with PB-CA-rtTAhygro 11 and CAG-PBase (Addgene, Watertown, USA) using the electroporator CUY21Pro-Vitro (Nepa gene, Chiba, Japan).Cells displaying resistance to both 100 μg/mL neomycin and 50 μg/mL hygromycin were assessed for inducible transgene expression upon treatment with 5 μg/ml dox and subsequently mixed for subsequent experiments.The transient expression of the transgenes in Cos7 cells was evaluated using Lipofectamine 2000 (Invitrogen-Thermo Fisher).

Preparation of recombinant GFP-mesotrypsin protein
To construct a bacterial expression plasmid for the production of recombinant GFP-mesotrypsin protein, cDNA encoding linker-mesotrypsin was ligated to the C-terminal sequence of 6 × Histidine tagged-GFP and subsequently inserted into the pet 3a plasmid (Sigma-Aldrich), yielding pet 3a-GFP-mesotrypsin.For control purposes, an analogous plasmid devoid of mesotrypsin cDNA was also generated (pet3a-GFP).The bacterial strain BL21 was transformed with either of these plasmids, cultured at 32 °C, and induced with isopropyl β-d-1-thiogalactopyranoside (IPTG) at 32 °C for 2 h.The resultant recombinant protein within the bacterial cells was purified utilizing NI-NTA columns (Qiagen, Hilden, Germany) and subsequently dialyzed against PBS.Both recombinant proteins yielded more than 1 mg/mL.The purified proteins were bound to NI-NTA beads (Qiagen) again and incubated with HaCaT cell lysate extracted in TBS containing 1% Triton X-100 for 1 h.The cell lysate was then analyzed for Pro-FLG.

Immunodetection
Western blotting was performed following established protocols, employing ECL reagent (Invitrogen-Thermo Fisher) for detection and LAS-500 (GE HealthCare, Chicago, USA) for analysis.When analyzing multiple proteins in a blot, protein-transferred membranes were cut into sections containing the target protein bands before incubation with antibodies (Thus, we cannot present full membranes for some samples; see Supplementary Fig. S4).Immunocytochemistry was performed as follows: Cells were cultured on a chamber slide (SPL Life Science, Gyeonggi-do, Korea) precoated with collagen and fixed with 4% paraformaldehyde in Tris-buffered saline (TBS) for 10 min.Subsequently, the cells were permeabilized with 0.1% Triton X-100 for 10 min, incubated with 4% skim milk in TBS (STBS) for 1 h, and incubated with primary antibodies in STBS for 2 h.Following TBS washing, the cells were treated with labeled secondary antibodies and counterstained with Hoechst 33258 www.nature.com/scientificreports/(Dojindo, Kumamoto, Japan).Analysis of the samples was conducted using a TCS SPE system (Leica, Wetzlar, Germany).The primary antibodies utilized in this study included antibodies against β-actin (Sigma-Aldrich), TGase-1 (Proteintech, Rosemont, USA), E-cadherin (ECCD2, a gift from Dr. Takeichi), LaminA/C (Cell Signaling, Danvers, USA), Lamin B1 (Abcam, Cambridge, UK), Rb, phosphorylated Rb (Cell Signaling Technology, Danvers, USA), ZO-1 (a gift from Dr. Nagafuchi), T7-tag (MBL, Tokyo, Japan), Ki67 (Abcam), Pro-FLG 12 , and GFP/Venus (culture supernatant of JFP-J1 cells, Riken Cell Bank RCB2309).Secondary antibodies conjugated to Alexa488, Cy3, or horseradish peroxidase (HRP) were obtained from Sigma-Aldrich and Merck-Millipore (Darmstadt, Germany).To quantify the accumulation of ZO-1 or E-cadherin, cells were stained for these molecules and photographed using a TCS SPE system (Leica, Wetzlar, Germany).A closed line surrounding each cell was then traced, and the total signal intensity was determined using the ImageJ system 55 .

Cell proliferation assay
In addition to analyzing Ki67-positive cells, we further evaluated the cell growth potential of the cells utilizing the Alamar Blue reagent.The cells were cultured in 96-well plates and exposed to 10% Alamar Blue (Invitrogen-Thermo Fisher) in DH10 medium.After incubating at 37 °C for 1 h, the absorbances at 570 and 600 nm were quantified using a Multiskan FC microplate reader (Invitrogen-Thermo Fisher), and the ratio of the change to the control was subsequently calculated.

Evaluation of cadherin-mediated cell adhesion strength
To assess the robustness of cadherin-mediated intercellular adhesion, a cell aggregation assay was conducted utilizing cells expressing only cadherins as intercellular adhesion molecules, as previously described 56 .The cells were treated with 0.01% trypsin in HEPES-buffered saline containing 2 mM CaCl2, which effectively eliminated most adhesion molecules while preserving cadherins 56 (Supplementary Fig. S3).Subsequently, the cells were thoroughly dissociated through vigorous pipetting, centrifuged, and reconstituted in a DH10 medium.After rotation at 100 rpm for 60 min, the size of the newly formed cell aggregates, which reflects the adhesive strength of the cadherin, was quantified.

Statistical analyses
The data are presented as the mean ± SD from a minimum of three independent experiments.Student's t-test was used to determine the p values, with values less than 0.05 considered to indicate statistical significance.In cases involving three or more samples, multiple testing was conducted using one-way ANOVA and the Tukey test.

Figure 1 .
Figure 1.Expression of Pro-FLG and its processing protease mesotrypsin in NHEK and HaCaT cells.(A) Morphological characterization of model cells.NHEK cells were treated with (+ Ca 2+ ) or without (− Ca 2+ ) 2 mM CaCl 2 for 2 days.Right, HaCaT cells cultured in DH10 medium.NHEK cells subjected to Ca 2+ treatment undergo differentiation and exhibit a flattened morphology.Scale bars, 200 µm.(B) Expression of Pro-FLG and its processing enzyme mesotrypsin.The Pro-FLG protein is abundantly expressed in both differentiated NHEK and HaCaT cells.(C) Analysis of the mRNA expression of the mesotrypsinogen gene (PRSS3) revealed that HaCaT cells demonstrate the considerably lower transcriptional activity of the mesotrypsin gene compared to NHEK cells.n = 4, *p < 0.05.There are significant variations in error bars among NHEK cell samples, attributed to substantial differences in the basal expression of PRSS3 in NHEK (lot 1) and NHEK (lot 2).(D) The expression of PRSS3 was evaluated in each experiment, elucidating that its upregulation associated with differentiation was exclusively observed in NHEK (lot 2).

Figure 2 .
Figure 2. The regulation of mesotrypsin expression in HaCaT cells is governed by a protein elimination system.(A) Schematic representation delineating doxycycline-inducible expression constructs for active mesotrypsin (left) and subsequent analyses of the transgene product in Cos7 or HaCaT cells (right).TRE, tetracycline/doxycycline-responsive element.T7-tagged active mesotrypsin was successfully generated in Cos7 cells, while its production was notably absent in HaCaT cells, despite its mRNA being expressed.n = 3, **p < 0.01.(B) Upper, Presentation of the expression construct featuring a Venus-mesotrypsin fusion protein linked by a peptide linker and TEV recognition site.Lower, Assessment of the exogenous Venus-mesotrypsin fusion protein in both Cos7 and HaCaT cells.Cos7 cells exclusively express the fusion protein (~ 60 kDa), whereas HaCaT cells produce an additional 38 kDa protein, recognized by anti-Venus/GFP antibodies, following doxycycline treatment.Antibodies targeting the T7 tag associated with mesotrypsin were used to identify the 60 kDa fusion protein, but the protein was not reactive toward the ~ 25 kDa Venus-free mesotrypsin in HaCaT cells.(C) Treatment with the proteasome inhibitor MG132 effectively prevented the time-dependent reduction in the Venus-mesotrypsin fusion protein (60 kDa).n = 3, *p < 0.05.

Figure 4 .
Figure 4. Mesotrypsin induces a flattened morphology and growth arrest in HaCaT cells.(A) Phenotypic depiction of HaCaT cells expressing Venus-mesotrypsin (right) and those without expression (left).Scale bars, 200 µm.(B) Left, Quantitative assessment of cell size.The expression of Venus-mesotrypsin, as opposed to Venus alone, results in a significant increase in cell size.Middle and right, Determination of cell thickness using z-stack images.n = 3, **p < 0.01.***p < 0.001.Randomly selected 20 cells were analyzed in each experiment.In contrast to Cos7 cells, HaCaT cells exhibit a flattened morphology upon Venus-mesotrypsin expression.(C) The Alamar Blue assay revealed growth arrest in HaCaT cells expressing Venus-mesotrypsin.n = 5, ***p < 0.001.

Figure 5 .
Figure 5. Mesotrypsin enhances adherens junctions (AJs).(A) Assessment of E-cadherin mRNA (left, n = 3) and protein (right, n = 3, **p < 0.01) expression.In addition, HaCaT cells expressing Venus-mesotrypsin exhibited upregulated E-cadherin.(B) Upper left, A schematic representation of the cell aggregation assay designed to evaluate cadherin activity.Cells subjected to trypsin treatment in the presence of calcium (TC treatment) eliminate most cell adhesion molecules, leaving cadherins intact.Disrupted cells in rotational culture reassembled into aggregates, the size of which reflects the activity of E-cadherin.Middle and lower, Representative images of reaggregated cells.Scale bars, 200 µm.Right, Quantification of aggregate sizes.n = 3, *p < 0.05.The expression of Venus-mesotrypsin, as opposed to Venus alone, results in the formation of large aggregates, indicating heightened cadherin activity.

Figure 6 .
Figure 6.Mesotrypsin plays a supportive role in forming adherens junctions (AJs) and tight junctions (TJs).(A) The experimental timeline for the analysis of TJ formation is depicted at the top.Immunolocalization of E-cadherin (green) and ZO-1 (red) in HaCaT cells expressing Venus only (left) or Venus-mesotrypsin (right).In the middle, HaCaT cells without transgene expression were generated.The cells were counterstained with Hoechst 33258.Scale bars, 50 µm.(B) Quantification of the accumulation of E-cadherin (left) and ZO-1 (right) in HaCaT cells expressing Venus-mesotrypsin.The signal intensities of E-cadherin (left) and ZO-1 (right) per unit length of the cell-edge lines were measured.n = 3, **p < 0.01.Randomly selected 30 (left) or 29 cells (right) were analyzed in each experiment.The presence of Venus-mesotrypsin significantly accelerates the accumulation of E-cadherin and ZO-1 at cell-cell contact sites.Treatment with only the JNK inhibitor was insufficient for promoting TJ formation in HaCaT cells expressing Venus alone or lacking Venus-mesotrypsin. https://doi.org/10.1038/s41598-024-63271-w