Extracellular epimorphin impairs expression and processing of profilaggrin in HaCaT keratinocytes

The expression and processing of filaggrin, a filament-associated protein in the skin epidermis, is closely associated with keratinocyte cornification. The large precursor profilaggrin (Pro-FLG) is initially detected at the granular layer in keratohyalin granules, subsequently processed into 10 to 12 filaggrin monomers (mFLGs) for keratin assembly, and ultimately degraded into smaller peptides that behave as natural moisturizing factor (NMF) at the outermost epidermis. We previously reported that epimorphin (EPM) extruded upon external stimuli severely perturbs epidermal terminal differentiation. Using HaCaT keratinocytes with inducible expression and recombinant EPM and FLG, we investigated the effect of extracellular EPM on the expression profile of filaggrin. As expression and processing of Pro-FLG in primary keratinocytes are accompanied with apoptotic cell death, we employed HaCaT keratinocytes that grow and express filaggrin mRNA in standard culture medium. In response to ectopic stimulation with extracellular EPM, Pro-FLG expression decreased with elimination of keratohyalin granules in the cells, with filaggrin mRNA remained constant and profilaggrin processing was not accelerated. Additionally, using a recombinant form of mFLG engineered for intracellular localization, we found that extracellular EPM hindered proteolytic cleavage of mFLG for production of NMF. Taken together, extracellularly extruded EPM, an epidermal cornification blocker, not only decreases Pro-FLG expression but also reduces the production of NMF in HaCaT keratinocytes.


Introduction
In the skin, undifferentiated keratinocytes in the epidermal basal layer continuously deliver daughter cells upward to give rise to a multilayered epidermis, and keratinocytes in each layer asynchronously produce a set of specific regulatory proteins to determine cell shape and differentiation state. Among these proteins, filaggrin is unique, in it is produced and actively processed upon epidermal terminal differentiation, and each processing product, as well as its large precursor protein profilaggrin (Pro-FLG), are endowed with distinct functions in different epidermal upper layers (Hoober and Eggink 2022). For example, in the granular layer, Pro-FLG, which comprises more than 4000 amino acids, is expressed as a major component of keratohyalin granules, then processed into N-terminal fragments and closely resembles 10 to 12 filaggrin monomers (mFLGs) in response to subsequent differentiation (Harding et al. 2013). The N-terminal fragment is reportedly imported into nuclei (Aho et al. 2012;Yamamoto-Tanaka et al. 2014), and mFLGs remaining at cytoplasm assemble the keratin intermediate filaments (Sumitomo et al. 2019), both of which prime programmed cell death. Upon terminal differentiation, mFLGs are further cleaved by caspase-14 (Eckhart and Tschachler 2011;Hoste et al. 2011) into small peptides that function as natural moisturizing factors (NMF) in the outermost epidermal cell layer (Harding et al. 2013). While the expression and processing of filaggrin are strictly controlled by phosphorylation and several molecular elements in accordance with epidermal differentiation and inflammation (Nakahara et al. 2021;Ovaere et al. 2009), the whole picture of their stimuli-related regulation remains unclear.
Epimorphin (EPM) which usually mediates intravesicular fusion as a t-SNARE protein, often translocates across the cell membrane in response to several external stimuli, such as toxic agents, hormones, and calcium influx, followed by its cleavage at the membrane-proximal domain for secretion (Hirai et al. 2007;Radisky et al. 2009). The extracellularly presented EPM executes its latent extracellular functions for epithelial morphogenesis and differentiation (Hirai et al. 2007;Radisky et al. 2009). In the skin epidermis, extracellular EPM appeared to severely perturb the differentiation program in keratinocytes and attenuate epidermal cornification (Okugawa and Hirai 2008). Although primary keratinocytes proceed cornification in response to high calcium, they quickly succumb to apoptosis, which would be problematic to assess the filaggrin processing (Norsgaard et al. 1994). Therefore, we analyzed HaCaT keratinocytes, which can be grown in typical culture medium with abundant expression of filaggrin mRNA (Wilson 2014), for the effect of extracellular EPM on the expression and processing of Pro-FLG. We detected important regulatory functions for the expression and processing of filaggrin in extracellularly extruded EPM.

Immunodetection
Immunoblot analysis was performed according to standard procedures. For immunocytochemistry, cells were cultured on 4-well chamber slides (SPL Life Sciences, Gyeonggi-do, Korea), fixed with 4% PFA in Tris-buffered saline (TBS) for 10 min and permeabilized with 0.1% Triton X-100 for 10 min. Each sample was then incubated with 3% skimmed milk in TBS at room temperature for 1 h, primary antibody for 15 h, and secondary antibodies for 1 h, followed by extensive washing with TBS at each interval. The nuclei were counterstained with DAPI (Sigma-Aldrich, MO, USA). Samples were analyzed using a Leica TCS SPE system (Leica Camera AG, Wetzlar, Germany).

Quantitative RT-PCR
The total RNA was extracted using a Total RNA Extraction Miniprep System (Viogene, CA, USA) and reverse-transcribed using an RNA-PCR kit (Takara Bio Inc., Otsu, Japan). Quantitative real-time PCR (qRT-PCR) was performed using the Fast Start Essential DNA Green Master on a Right Cycler Nano system (Roche Diagnostics, Mannheim, Germany). The cDNAs were amplified for filaggrin, exogenous T7-tagged epimorphin, KLK5, LEKT1, Calpain-1, and GAPDH genes using primer pairs listed in Table 1 The relative expression of mRNA was normalized to that of GAPDH.

Statistical analyses
Data are presented as the mean ± standard deviation from at least three independent experiments. The p-values were determined by Student's t-test, and p-values less than 0.05 were considered statistically significant.

Expression of filaggrin in HaCaT keratinocytes
We initially generated recombinant filaggrin monomers containing a linker domain, r-mFLG1 and 2 (Supplementary Fig. 1), which behaved as a 45 and 42 kDa protein, respectively (Fig. 1a), despite the estimated masses being around 34 kDa, which was consistent with a previous report using the equivalent r-mFLG (Hoste et al. 2011). Polyclonal antibodies against mFLG1 selectively labeled upper layers of the skin epidermis, consistent with filaggrin distribution (Fig. 1b). To investigate the expression profile of filaggrin, we employed HaCaT keratinocytes cultured in conventional medium, since these cells do not undergo apoptosis with abundant expression of filaggrin mRNA (Wu et al. 2018), retain several keratinocyte characteristics (Boukamp et al. 1988), and are readily transfected (Fusenig and Boukamp 1998;Okugawa and Hirai 2008). The antibodies could predominantly detect the large proteins in HaCaT cells, but not those with genetic ablation of filaggrin, demonstrating that HaCaT cells produce profilaggrin (Pro-FLG). On the other hand, apparent signals for mFLGs (30-45 kDa) were not detected, and the artificial expression of the gene product of introduced mFLG1 was not feasible in HaCaT cells, albeit Cos-7 cells produced small amount of 37 and 30 kDa proteins (Fig. 1c). These results indicate that HaCaT keratinocytes lost the ability to process Pro-FLG, or mFLGs are unstable in these cells (Fig. 1b  and c), albeit they were ascertained for the downregulation of several differentiation markers including Pro-FLG in low Ca 2+ medium (data not shown).
Given that Pro-FLG-processed products, mFLGs and the N-terminal fragment, prime apoptotic cell death via assemble keratin cytoskeleton (Kuechle et al. 2000;Presland et al. 2001) or intranuclear components (Ishida-Yamamoto et al. 1998), these results might explain why HaCaT cells can be maintained in conventional medium with relatively higher Ca 2+ . Also, it is suggested that the effective processing of Pro-FLG does not occur in cultured keratinocytes (Pearton et al. 2002).

EPM and expression of pro-FLG
We previously showed that extracellular epimorphin (EPM) prevented cornification in HaCaT cells (Okugawa et al. 2010). To address its possible involvement in the expression of Pro-FLG, these cells were introduced with an expression construct for extracellular EPM tagged with the T7-peptide under the control of dox (Fig. 2a). Treatment with dox resulted in the expression of T7-tagged extracellular EPM (35 kDa and the glycosylated 37 kDa form), as has been previously shown (Hirai et al. 1998). Subsequently, these cells significantly decreased the amount of Pro-FLG (Fig. 2b), and keratohyalin granule-like structures comprising Pro-FLG were almost eliminated in cells those with high EPM expression (Fig. 2b). Intriguingly, filaggrin mRNA was not downregulated in cells with EPM (Fig. 2b), and this was the case for several pro-FLG-processing proteases (Lin et al. 2020;Resing et al. 1993;Sakabe et al. 2013;Yamazaki et al. 1997) including calpain-1, kallikrein-5 (KLK5), and KLK5-regulator LEKT1 (Supplementary Fig. 2a). Additionally, artificial inhibition of proteasomal or lysosomal Fig. 1 Preparation of specific antibodies against filaggrin monomer (mFLG). a upper left, schematic diagram of Pro-FLG and recombinant mFLG proteins. Primer pairs for mFLG amplified two different amplicons, mFLG1 and mFLG2. Lower, amino acid sequence of 6X Histidine-tagged forms of recombinant mFLG1 (r-FLG1). mFLG1 and mFLG2 are more than 95% identical to sequences in public database (see Supplementary Fig. 1), and retain consensus sequences for caspase-14 recognition (Blue). Mutated amino acids harboring the isolated mFLG1 are shown in red. Upper right, SDS-PAGE and western blot analyses of purified r-mFLG1 and r-mFLG2. CBB, Coomassie Brilliant Blue. Anti His-tag, proteins detected by antibodies against His-tag antibodies. Lower protein bands, 23 kDa in mFLG1 and 21 kDa in mFLG2, might represent the degraded products lacking C-terminus. b left, cryosections of mouse dorsal skin stained with polyclonal antibodies generated using r-mFLG1 (Abs1) or r-mFLG2 (pAb2    Fig. 2b and c), suggesting that the decrease in Pro-FLG expression might be attributed to perturbation of protein translation. Indeed, while phosphorylation of the translation initiation factor elF4E was not affected, an active phosphorylation of ribosomal protein S6 (RPS6), which reportedly occurred when translation of mRNA having long ORF is suppressed (Bohlen et al. 2021), was seen in cells expressing EPM (Fig. 2c). That these translational regulators are known as downstream elements of mTOR (Ruvinsky and Meyuhas 2006) and phosphorylation was clearly enhanced only in RPS6, suggests that RPS6 activity is regulated by non-canonical pathways. It is notable, however, that doxycycline reportedly exerts unexpected functions in addition to antibacterial actions in keratinocytes (Ishikawa et al. 2009;Kanada et al. 2012;Lindsay et al. 2008;Uitto et al. 1994). To further ascertain the effect of extracellular EPM on the attenuation of Pro-FLG, parental HaCaT cells were treated with active form of recombinant EPM (r-EPM), in the absence of dox. Consistent with the results using HaCaT transfectants, r-EPM decreased Pro-FLG levels in the cells (Fig. 2d). Thus, it is conceivable that signals propagated by extracellular EPM hinder the translation of filaggrin mRNA (Fig. 2e).
EPM and processing of mFLGs Next, we tried to investigate possible effects of extracellular EPM on the cleavage of mFLGs that occurs upon epidermal cornification. As seen in transient mFLG expression (Fig. 1c), HaCaT cells failed to express exogenous mFLG1 by a stable expression system using piggyback transposon activity (Fig, 3a). In addition, it has been suggested that doxycycline might regulate enzymatic activity of caspase 14, a keratinocyte-specific protease responsible for mFLG cleavage (Lippens et al. 2000;Yamamoto-Tanaka and Hibino 2014). Thus, we used another approach where recombinant mFLG1 was fused with a cellpenetrating peptide (Stout et al. 2014), and added to HaCaT cells treated with r-EPM. To this end, we used a fusion protein system established by Liu laboratory , where flag-tagged mFLG1 was fused with positively-charged GFP (+ 36GFP) for cell surface association followed by endocytotic incorporation, and an antibacterial peptide, Aurein, for disruption of the endosomal membrane (r-A/36G/flag-mFLG1) (Fig. 3b). While r-GFP did not adsorb to the surface of HaCaT cells, r-A/36G/flag-mFLG1 was effectively incorporated and accumulated in the cytoplasm (data not shown, see Fig. 3c). Analyses using anti-flag tag antibodies revealed that r-EPM protects degradation of mFLG1 (Fig. 3c), coincidently with the tendency to prevent cleavage/activation of cas-pase14, a mFLG-cleavage enzyme for NMF production (Eckhart and Tschachler 2011;Hoste et al. 2011) (Fig. 3d). Concomitant with the inhibitory effect on the expression of Pro-FLG, a source of mFLGs (as shown in Fig. 2), these results indicate that the signals propagated by extracellular EPM decreases the amount of NMF in fully differentiated keratinocytes. Considering that decline in NMF is a typical symptom of atopic dermatitis (Kezic et al. 2008), the elucidation of the molecular mechanism underlying EPMdependent regulation of Pro-FLG and mFLGs might provide clinical benefit.

Conclusion
This study revealed that EPM, which is extruded upon several external stimuli, not only decreases the expression of Pro-FLG but also hinders cleavage of mFLGs for NMF production (Fig. 4). At present,

Pro-caspase-14
Active (  shown to behave as cell death/tumorigenic drivers to cause morphological abnormality in the mouse mammary epithelia (Bascom et al. 2005;Hirai et al. 2001). Intriguingly, the mammary epithelial cells are known to express FLG upon undergoing anoikis or oncogenic transformation (Beretov et al. 2015;Mailleux et al. 2007).
In conclusion, molecular elements for membrane translocation of EPM might be therapeutic targets of keratotic lesions, considering that EPM regulates expressions of Pro-FLG, mFLG and NMF in culture, all of which play distinct but critical roles in epidermal cornification in vivo.