INVESTIGATION OF SPHINGOSIN-1-PHOSPHATE-TRIGGERED MATRIPTASE ACTIVATION USING A RAT PRIMARY HEPATOCYTE MODEL

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The major internal detoxification organ in mammals is the liver, which serves a wide spectrum of vital functions.Liver cells are divided into hepatocytes, Kupffer cells, and endothelial cells.Transformed hepatocyte cell lines, for example rat hepatoma H-4-II-E cells (Kovacs et al., 1977) cannot adequately mimic the phenotype of the liver.In contrast, the primary cells were isolated directly from the tissues and therefore have a normal cell morphology and are ca-pable of maintaining many of the markers and functions seen in vivo (Pan et al., 2009).The rat primary hepatocyte model is an important tool for hepatic toxicology studies.However, it is well reported that primary cells have a limited lifetime (2-4 days) (Soldatow et al., 2013).
Matriptase-1 (MT-1, also known as epithin) and matriptase-2 (MT-2, also known as TMPRSS6) belong to the type II transmembrane serine protease family (TTSP) (Hooper et al., 2001), more precisely to the matriptase subfamily (Szabo and Bugge, 2008).MT-1 is widely expressed in epithelial tissues, with high levels in the gastrointestinal tract, prostate (Takeuchi et al., 1999), normal and cancerous breast, ovarian and colon tissues (Oberst et al., 2003).MT-1 is required for postnatal survival.According to Oberst et al. (2001), MT-1 has a potential role in cancer invasion and metastasis in some types of cancer.
MT-2 has been found in high levels in the liver and in small amounts in the kidney (Hooper et al., 2003).MT-2 is structurally similar to MT-1 (Sanders et al., 2010).Silvestri et al. (2008) identified the membrane-bound haemojuvelin as a substrate for MT-2 in vitro.MT-2 can regulate the iron homeostasis (Folgueras et al., 2008).Hepcidin is a systemic iron regulatory hormone, secreted by the liver (Du et al., 2008).It modulates the iron absorption via duodenal enterocytes (Nicolas et al., 2001).Hepcidin negatively regulates the iron export by promoting the degradation of ferroportin, which is an iron exporter located on the surface of intestinal enterocytes, macrophages, and hepatocytes (Nemeth et al., 2004).MT-2 cleaves membrane-bound haemojuvelin which is the main activator of hepcidin transcription (Silvestri et al., 2008).Dysfunction of matriptase-2 (MT-2) can be involved in iron regulatory disorders via altered regulation of hepcidin expression.
Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite.Most cells can synthesise S1P which is an intracellular second messenger and extracellular mediator in mammalian cells (Yatomi et al., 2001).S1P regulates a wide variety of biological responses, for example angiogenesis, vascular maturation, cardiac development and immunity, and is important for cell proliferation, cell survival and cell movement.There are five G-protein-coupled S1P receptors which enable regulation of numerous downstream signalling pathways.The role of S1P depends on the relative expression of these receptors (Spiegel and Milstien, 2003).Benaud et al. (2002) determined a novel biological function of S1P as an activator of matriptase in epithelial cells.It was also demonstrated that bioactive phospholipids can activate cell surface proteases (Benaud et al., 2002).
The aim of this study was to investigate the possible relationship between S1P and hepcidin production in a rat primary hepatocyte model.Firstly, we estimated the cytotoxic properties of S1P and then we also investigated the effect of S1P on baseline extracellular H 2 O 2 production.The changes in hepcidin levels in cell-free supernatants were also quantified.

Exposure of primary hepatocytes to S1P
S1P was purchased from Bio-Techne (Minneapolis, Minnesota, United States).S1P was dissolved in phosphate-buffered saline with 4 mg/ml bovine serum albumin, which was the stock solution (0.5 mg/ml).After the first 6 h, S1P was added to the medium apically at 50, 200 or 1000 ng/ml.The maintenance medium with S1P was changed every 24 h.The treatment time was 72 h.

MTS assay for cell viability
The hepatocytes were seeded in a 96-well plate and incubated with 50, 200 or 1000 ng/ml S1P for 72 h.The control wells were incubated with maintenance medium only.After the medium was removed, 100 µl maintenance medium was added with 20 µl CellTiter96 aqueous one solution (Promega, Bioscience, Hungary) in every well.This reagent contained the tetrazolium compound MTS (Cory et al., 1991).The assay protocol is based on the reduction of the MTS tetrazolium compound by viable cells to generate a coloured formazan dye that is soluble in cell culture media.This conversion is reported to be carried out by NAD(P)H-dependent dehydrogenase enzymes in metabolically active cells.The cells were incubated with the dye for 2 h at 37 °C, 5% CO 2 .The absorbance of formazan was measured by EZ Read Biochrom 400 microplate reader at 490 nm wavelength (Biochrom Ltd., UK).

Amplex Red assay for extracellular H 2 O 2 level measurement
Amplex Red Hydrogen Peroxide Assay Kit (Thermo Fisher Scientific, USA) was used to measure the concentration of H 2 O 2 in cell-free supernatant from the apical compartment.Fluorescence ROS measurement was based on the fact that Amplex Red reagent reacts with H 2 O 2 and horseradish peroxidase and it is converted into fluorescent resorufin.The cells were treated with 50, 200 or 1000 ng/ ml S1P for 72 h.The samples were collected every 24 h; 50 µl cell-free supernatant and 50 µl Amplex Red working solution was used to determine the H 2 O 2 concentration.After 30 min of incubation at room temperature the samples were measured with Victor X2 2030 fluorometer at 560 nm excitation and 590 nm emission wavelength.

Hepcidin sandwich ELISA for determination of hepcidin level
To determine the concentration of hepcidin in the apical compartment from the cell-free supernatant, we used a rat hepcidin ELISA kit from Elabscience Biotechnology (Houston, Texas, USA).After 24, 48 and 72 h of S1P treatment at 50, 200 or 1000 ng/ml concentrations, the apical cell-free supernatants were collected.The assay procedure was performed according to the manufacturer's guidelines.The absorbance values were detected with EZ Read Biochrom 400 microplate reader at 450 nm wavelength (Biochrom Ltd, UK).

Statistical analysis
Differences between groups were statistically analysed using one-way analysis of variance (ANOVA) with Tukey's post hoc test, where the data were of normal distribution and the homogeneity of variances was confirmed.Statistical significance was set at P < 0.05.For statistical evaluation the R 2.14.2 software package was applied (2012, Vienna, Austria).The results are expressed as means ± SDs.

The effect of S1P on cell viability
It was confirmed that even the highest concentration of S1P (at 1000 ng/ ml) did not cause significant cell death after 24-h (P = 0.96887) and 72-h (P = 0.1960) treatment (Fig. 1).It was found that S1P administration for 72 h even at 1000 ng/ml was safe in the case of the rat primary hepatocyte model.

BARNA et al.
Acta Veterinaria Hungarica 67, 2019 Fig. 1.Assessment of cell viability after 24 and 72 h of incubation of rat primary hepatocytes with S1P at 50, 200 and 1000 ng/ml.The results represent average absorbance values of produced MTS formazan ± standard deviations.No significant differences were found between control and S1P-treated cells after 24 and 72 h of incubation.The results are representative of three independent experiments

The assessment of H 2 O 2 measurement
We found that S1P did not cause a significant increase in the extracellular H 2 O 2 levels at any applied concentrations within the investigated time period (P > 0.05 in each case) (Fig. 2).

S1P-caused changes in hepcidin levels
It was found that the hepcidin levels in cell-free supernatants were significantly decreased compared to control samples after 24 h of incubation at each concentration of S1P (at 50 ng/ml and 200 ng/ml: P < 0.001, at 1000 ng/ml: P < 0.01).Hepcidin production was significantly reduced when cells were treated with S1P at 200 ng/ml (P = 0.0206) and 1000 ng/ml (P < 0.001) after 48 h of incubation (at 50 ng/ml: P = 0.8656).After 72-h administration of S1P, there were significant differences in hepcidin levels between the treated and the control samples (in the case of S1P at 50 ng/ml: P = 0.00124, in the case of S1P at 200 ng/ ml and at 1000 ng/ml: P < 0.001) (Fig. 3).
Fig. 3. Absorbance values of hepcidin in rat primary hepatocytes exposed to S1P for 24, 48 and 72 h.The hepcidin levels in the cell-free supernatant were significantly decreased after 24 h of incubation at all concentrations of S1P (50 ng/ml and 200 ng/ml: P < 0.001, 1000 ng/ml: P < 0.01).

Discussion
MT-1 has an important role in regulating the formation and integrity of the intestinal epithelial barrier (Buzza et al., 2010), development of the epidermis, hair follicles, and cellular immune system, and it is required for postnatal survival (List et al., 2002) via tight junction regulation.S1P as an extracellular mediator and intracellular second messenger can activate MT-1 in nanomolar concentrations (Benaud et al., 2002).
In our earlier study, S1P was tested on IPEC-J2 non-tumorigenic cell line (Pászti-Gere et al., 2016).IPEC-J2 was isolated from the jejunum of a neonatal non-suckled piglet (Berschneider, 1989).The main advantages of IPEC-J2 cells were that they possess enterocyte-like microvilli and tight junctions (Schierack et al., 2006).Occludin, claudin-1, -3, -4, -5, -7, and -8 were also detected (Zakrzewski et al., 2013) in IPEC-J2 cells.Thus, the IPEC-J2 cell line more closely mimics in vivo conditions than other, tumorigenic cell lines (Vergauwen, 2015).In our pre- vious study it was found that S1P at 200 ng/ml concentration was not cytotoxic and did not cause an elevation in extracellular hydrogen peroxide levels.S1P had strong barrier protective effect, since the transepithelial electrical resistance was elevated and a higher amount of occludin appeared in cell membranes (Pászti-Gere et al., 2016).MT-2 regulates the iron homeostasis via cleavage of membrane-bound haemojuvelin, which is a main activator of hepcidin transcription (Silvestri et al., 2008).Hepcidin inhibits the entry of iron into the plasma and its absorption from enterocytes.It was also observed that mice with a TMPRSS6 -/-defect or the absence of matriptase-2 proteolytic activity have iron deficiency anaemia, upregulation in hepcidin transcription, and reduced protein levels of ferroportin on enterocytes (Du et al., 2008;Folgueras et al., 2008).Thus, stimulation of MT-2 activation could be an alternative treatment of iron deficiency anaemia.
Our aim was to elucidate the effects of S1P on MT-2 indirectly via monitoring hepcidin levels on rat primary hepatocytes.S1P regulates numerous downstream signalling pathways, for example cell proliferation, survival and cell motility (Spiegel and Milstien, 2003).The role of S1P was examined using several different cells.In the liver the modulation of S1P can be a potential therapeutic target for the treatment of several hepatic diseases.It was observed that sphingolipid metabolites produce a significant upregulation of long-chain ceramides in the serum of patients with hepatocellular carcinoma (HCC) as compared to patients with cirrhosis (Grammatikos et al., 2016).In contrast, S1P (between concentrations of 10 and 30 µM) reduced the HCC cell migration induced by hepatocyte growth factor in HuH7 cells (Matsushima-Nishiwaki et al., 2018).In mice, the S1P level and the expression of one of its receptors (S1P3) in the liver were up-regulated in cholestasis-induced liver fibrosis (Li et al., 2009).Two animal models of type 1 diabetes had elevated S1P levels compared to control mice (Fox et al., 2011).However, the role of S1P in hepatic dysfunctions has not been completely clarified yet.
On the one hand, S1P could elevate H 2 O 2 level in FRTL-5 thyroid cells (Okajima et al., 1997), while on the other hand Nakahara et al. (2012) described that S1P can inhibit the apoptosis of granulosa cells.
Rat primary hepatocytes represent the in vivo conditions better than a hepatoma cell line, therefore the modulating effect of S1P on matriptase activity can be better predicted.According to our findings, S1P, in the tested concentration range, did not affect cell viability and extracellular H 2 O 2 levels significantly.Based on our results, the hepcidin levels significantly decreased in rat primary hepatocytes exposed to S1P after an incubation time of 24, 48 and 72 h.
In conclusion, the application of S1P seems to be safe at 50, 200 and 1000 ng/ml concentrations, because it did not increase the cell death rate of hepatocytes and did not disturb redox cell status.S1P treatment led to decreased hepcidin production; however, the changes in hepcidin levels were not fully de-pendent on S1P concentration.The results of this study indicate the beneficial properties of S1P in the treatment of disturbed iron homeostasis.

Fig. 2 .
Fig. 2. The effects of S1P at 50, 200 and 1000 ng/ml on extracellular H 2 O 2 production of rat primary hepatocytes.Cells were exposed to S1P for 24, 48 and 72 h.No significant differences were found between the control and the S1P-treated groups.The values represent the means of resorufin fluorescence intensities ± standard deviations (n = 3)