Pulmonary fibrosis in vivo displays increased p21 expression reduced by 5-HT2B receptor antagonists in vitro – a potential pathway affecting proliferation

Serotonin (5-hydroxytryptamine) has repeatedly been associated with the development of fibrotic disorders such as pulmonary fibrosis. By blocking the binding of 5-HT to 5-HT2B receptors with receptor antagonists, several pro-fibrotic mechanisms can be inhibited. Bleomycin-induced pulmonary fibrosis is a model used to evaluate pathological mechanisms and pharmacological interventions. Previously we have shown attenuated fibrosis in systemic bleomycin-treated mice following treatment with two 5-HT2B receptor antagonists (EXT5 and EXT9). Our aim is to further identify cellular effects and signaling pathways associated with the anti-fibrotic effects of EXT5/9. Gene expressions in lung tissues from systemic bleomycin-treated mice were examined, revealing significant increased expression of Cdkn1α (a gene coding for p21), particularly in distal regions of the lung. In vitro studies in human lung fibroblasts revealed increased levels of p21 (p = 0.0032) and pAkt (p = 0.12) following treatment with 5-HT (10 µM). The induction of p21 and pAkt appears to be regulated by 5-HT2B receptors, with diminished protein levels following EXT9-treatment (p21 p = 0.0024, pAkt p = 0.15). Additionally, 5-HT induced fibroblast proliferation, an event significantly reduced by EXT5 (10 µM) and EXT9 (10 µM). In conclusion, our results suggest that 5-HT2B receptor antagonism attenuates pulmonary fibrosis in part by anti-proliferative effects, associated with inhibited pAkt/p21 signaling pathway.

Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial lung disease with unknown etiology and limited treatment options 1 . The pathogenesis of IPF resides in ongoing and aberrant chronic wound repair responses. Realistic models of this disease are lacking, but the anti-tumor drug, bleomycin (BLM) is widely used to model pulmonary fibrosis in vivo via subcutaneous (s.c.), i.e. systemic administration, or more commonly through an intratracheal route of administration 2,3 . We have previously shown that following systemic BLM administrations, animals display increased accumulation of collagens, extracellular matrix (ECM) proteins and collagen producing cells 3,4 , mimicking numerous pathological mechanisms associated in IPF 5 . In response to lung injury and tissue repair, several molecular mechanisms are triggered such as proliferation of fibroblasts and the formation of collagen-producing myofibroblasts 6 , resulting in selective gene expressions. The formation of collagens correlates with upregulation of genes involved in cell proliferation 7 , underlining the important interplay between ECM deposition and cell proliferation. Furthermore, in BLM-treated mice, pulmonary levels of serotonin (5-hydroxytryptamine, 5-HT) and expression of 5-HT 2 receptors are elevated 8,9 , which is believed to be associated with increased myofibroblast differentiation and matrix deposition. This is supported by findings showing that treatment with selective 5-HT 2 receptor antagonists inhibit BLM-induced tissue remodeling 4,10 .
Several important cellular responses are mediated via 5-HT 2 receptors. In human endothelial cells, 5-HT has been shown to trigger an increased expression of Akt, a serine/threonine protein kinase involved in signaling pathways regulating migration, survival and proliferation 11 . Phosphorylated Akt (pAkt) and 5-HT as well as the expression of the 5-HT 2B receptor were increased in lungs of mice following orotracheal administered BLM 8 . One phosphorylation substrate for pAkt is p21 12 , a protein elevated at cellular damage and in pulmonary fibrosis [13][14][15][16] . Akt also promotes cell cycling by regulating substrates such as p21 and cyclin D1. Akt-induced p21 can promote the assembly and activation of cyclin D1-Cdk4 complex, triggering G1/S transition 17,18 . 5-HT modulates many important regulatory functions, and a recent study demonstrates increased cell proliferation and DNA synthesis in hepatocytes following activation of 5-HT 2B receptors 19 .
We recently described how a preventive treatment with 5-HT 2B receptor antagonists EXT5 and EXT9 inhibited several pulmonary fibrotic events in BLM-treated mice, such as reduced numbers of pulmonary myofibroblasts and collagen producing cells as well as an overall reduced pulmonary tissue density 4 . In the present study, we continued to investigate the effect of EXT5 and EXT9, focusing on pulmonary gene expressions in BLM-treated mice, suggestive of signaling pathways associated with cell proliferation. The effects of the 5-HT 2B receptor antagonists on proliferation and related intracellular signaling proteins were further studied in human in vitro cultures, relating to our earlier anti-fibrotic findings in vivo 4 .

Results
Gene for p21 upregulated in BLM-treated mice. We have previously examined initial events in pulmonary fibrosis in vivo, utilizing a formerly described model with repeated s.c. administrations of BLM -a systemic model demonstrating early alterations in ECM lung compositions and cell turnover 3,4 . Whole genome gene expression in distal lung tissue was analyzed after two weeks of BLM-treatment. Eight probes, coding for six genes, were significantly differentially expressed in BLM-treated mice, in comparison to control animals (Fig. 1). The expression of Cdkn1α was significantly upregulated (p < 0.05, q = 0) in BLM-treated mice, recognized by multiple significant probes for Cdkn1α (Table 1). Cdkn1α codes for p21, a protein associated with cell cycle regulation and cell damage 15 . Treatment with EXT9, and to some extent EXT5, showed a modest reduction in Cdkn1α expression, however the fold change (FC) of the probes was not proven as statistical significant, in comparison to BLM-treated mice. Two weeks of systemic administrations of BLM generated a mild to moderate model of pulmonary fibrosis, thus accompanied with small variation in disease related gene expressions. Analysis of lung tissue samples (examining larger parts of the lung) with rt-qPCR, demonstrated small differences in levels of Cdkn1α between treatment groups with some expression variability within groups (Fig. 2). In BLM-treated mice a minor increase of Cdkn1α (ΔCt = 5.48, FC = 1.1) was detected, in comparison to control animals (ΔCt = 5.60, FC = 1). Treatment with EXT5 or EXT9 showed tendencies in reducing the expression (EXT5 ΔCt = 6.07, FC = 0.7; EXT9 ΔCt = 5.75, FC = 0.9), however, not proven as statistical significant (Fig. 2).

Reduced proliferation of lung fibroblasts.
We have previously shown that collagen producing cells i.e.
Undetected cell toxicity with 5-HT 2B receptor antagonists. To exclude possible cytotoxic mediated effects, LDH release and cell viability were examined in human lung fibroblasts treated with 5-HT 2 receptor antagonists. After 48 h exposure to 10 µM of EXT5, EXT9 or RS 127445, HFL-1 cells displayed neither elevated levels of LDH (Fig. 5a), nor a reduction in viability with WST-1 (Fig. 5b). Results show that none of the tested compounds at the highest tested concentration (10 µM) caused cell-mediated cytotoxicity, with the application of a 5% cut-off.

Discussion
We have previously shown that an experimental induced model of pulmonary fibrosis, implementing systemic administrations of BLM, resulted in increased pulmonary tissue accompanied with increased amount of ECM-producing cells 4 . In this follow-up study, we identified an increased expression of Cdkn1α, a gene coding for the p21 protein, in BLM-treated mice. This protein, regulating cell growth was examined in human lung fibroblasts, serving as a translational study. Human lung fibroblasts demonstrated an induction of p21 following 5-HT-exposure, as well as the upstream signaling protein pAkt, which was reduced by treatment with 5-HT 2B receptor antagonists. Receptor mediated effects on cell proliferation and cell cycle entry showed a reduction in cellular growth, correlating to the recognized cell regulation pathways of pAkt/p21 12,18 . Following intratracheal (local) BLM administration, severe damage occurs in epithelial cells, which is followed by activation and proliferation of fibroblasts, and subsequently also fibrosis 23 . Active and proliferative fibroblasts are thought to be key players in fibrosis, causing excess deposition of matrix proteins and collagen. Transforming growth factor (TGF) -β1, is a pro-fibrotic mediator stimulating proliferation and differentiation of fibroblasts into myofibroblast 24 and upregulation of p21 25 ; a marker for cellular damage and regulator in cell cycle progression 12 . In patients with IPF, p21 is overexpressed in terminal airways and alveoli 15 , highlighting distal lung regions of chronic cellular damage, which is the focus of the current study. Our previous studies have demonstrated that systemic administrations of BLM induced pulmonary fibrosis, with increased tissue density, as well as increased collagen staining (Masson's trichrome and Picro Sirius red) and increased numbers of tissue myofibroblasts 3,4 . Systemic administrations of BLM initiates damage and stress to endothelial and epithelial cells, promoting the development of fibrosis in parallel with inflammation 3 . This mild to moderate model of pulmonary fibrosis, with low adverse effects, differs from the acute model of BLM-induced fibrosis using local administration. We thus wanted to investigate gene expression in distal lung tissue following systemic administration of BLM, seeking to identify signaling pathways involved in early fibroblast/myofibroblasts activation and proliferation. We identified Cdkn1α (gene coding for p21) to be significantly increased in BLM-treated mice in comparison to control animals (Table 1). These results supports previous findings in BLM-treated mice, which demonstrated an increased pulmonary expression of p21 positive cells 26 . A reduction in Cdkn1α with subsequently reduction of p21, may be indicative of a reduced cellular damage and less on-going repair process. Treatments with EXT5 or EXT9 showed tendencies in reducing the expression of Cdkn1α in comparison to BLM-treated mice, however, we were unable to confirm these results statistically. Comparisons of control animals to BLM-treated mice enable the identification of differential expressed genes, that showed tendencies in gene levels reverting back toward that of control animals following treatment with EXT, even though not significant. In our follow-up study examining both central and distal parts of the lung, similar expression patterns of Cdkn1α was seen in mice treated with EXT5 or EXT9, in comparison to BLM-treated mice. The heterogeneity of the disease model and usage of low systemic dosage of BLM may explain the small differences observed in between treatment groups. Origin of tissue is also crucial when studying this disorder, as the expression of Cdkn1α appears more prominent in parenchymal tissue. In summary, these two studies identify early changes in the gene expression of p21 in pulmonary fibrotic mice, with results indicative of 5-HT 2B receptor regulations.
The regulatory functions of p21 are highly complex, with several associated cellular mechanisms such as regulation of cell cycle progression and promotion of cell survival 17 . The nuclear export of cyclin D1 can also be inhibited by p21, thus promoting nuclear accumulation of cyclin D1, triggering cell cycling 27 . Cell cycle progression has been linked to 5-HT and 5-HT 2 receptor activation 21,28 . In mouse fibroblasts transfected with the 5-HT 2B receptor, activation of receptors resulted in elevated cyclin D1 expression and thus increased cell proliferation, which was decreased with 5-HT 2B receptor antagonism 21 . In line with these findings, we showed that 5-HT enhances the entry of lung fibroblasts into the dividing phases of cell cycling, an event clearly diminished with 5-HT 2B receptor antagonism.
Interestingly, reduced levels of pulmonary pAkt, 5-HT and 5-HT 2B receptors have been associated with an attenuated inflammation and fibrosis 8 . In bovine pulmonary artery smooth muscle cells, 5-HT as well as a 5-HT 2 agonist induced phosphorylation of Akt, an activation not hindered by the 5-HT 2B receptor antagonist -SB215505 28 . In this study we demonstrated anti-proliferative effects with 5-HT 2B receptor antagonists EXT5 and EXT9 in human lung fibroblasts, a non-toxic mechanism, previously shown to attenuate myofibroblast differentiation 4 . These results support a dual action of the 5-HT 2B receptor antagonists in inhibiting both cell differentiation and proliferation, however, distinguishing which cellular action is the foremost driving force in diminishing the development of fibrosis is still to be elucidated.
EXT5 and EXT9 have shown to display similar although varied effects in attenuating fibrosis in vitro and in vivo 4 , possibly explained by the compounds' different receptor binding and functionality profiles. In current study, no distinct difference was detected in anti-fibrotic effects elicited by the two compounds, hence, the slight variance in receptor profiles appears less relevant.
We previously showed that EXT5 and EXT9 decreased the production of the proteoglycans such as decorin 4 . Interestingly, several studies have identified the interplay between p21 and decorin, presenting decorin as an inducer of p21 29,30 . In our previous study, we identified the upregulation of decorin in lungs of BLM-treated mice, as well as in human lung fibroblast treated with 5-HT in combination with TGF-β1 4 . Decorin production has shown to be increased in fibroblast clones from IPF patients, however, identified with a negative correlation between decorin synthesis and cell proliferation 31 . In current study, results showed the induction of p21 by 5-HT (10 µM), as well as the induction of pAkt, supporting previous findings in bovine smooth muscle cells 28 . The induction of pAkt and p21 may be linked to amplified levels of decorin via activation of 5-HT 2B receptors, a theory supported by reduced decorin production following treatment with EXT5 and EXT9 4 . However, with several anti-fibrotic effects documented, the cellular and molecular mechanisms of 5-HT 2B receptors antagonism in pulmonary fibrosis warrants further investigation 4,9 . This study has focused on identifying early fibrotic events using a well characterized fetal cell line -a cell type linked to on-going regenerative processes in the development of fibrosis. In IPF patients 32 and in BLM-induced fibrosis 9 pulmonary expressions of 5-HT 2A and 5-HT 2B receptors are increased, whose expression on HFL-1 cells has been previously confirmed 4 . The anti-fibrotic effects mediated by EXT5 and EXT9 in vivo, translate to human cell culture systems, proposing the 5-HT 2B receptor antagonists as a novel approach in treating human pulmonary fibrotic disorders. For further clinical bearing, it would be of interest to examine a therapeutic treatment option with the compounds, when compound administration is applied later in the development of the disease. Studies have suggested 5-HT 2 receptors as potential targets of novel treatment options for several fibrotic conditions such as hepatic fibrosis, systemic scleroderma and pulmonary hypertension 33 , thus highlighting the wide potential of selective 5-HT 2 receptor antagonists in treating fibrosis of different tissue origin. In conclusion, the results from this study further identify the mechanism of 5-HT 2B receptors in associated pulmonary remodeling processesaffecting cell proliferation and associated signaling pathway pAkt/p21 both in vitro and in vivo.

Methods
Pulmonary fibrosis in vivo model. C57BL/6 mice (female, aged 12.5 weeks) (Scanbur research A/S, Karlslunde, Denmark) were injected s.c. with BLM three times/week for two weeks, with daily per oral (p.o.) treatment with EXT5 (30 mg/kg), EXT9 (30 mg/kg) or vehicle (Tween80, 2.5%w/v) as previously described 4 . The administration of compounds and vehicle were given simultaneously with first dose of BLM. Control animals were injected s.c. with saline, followed by p.o. treatment with vehicle. Seven animals were used per treatment/ control groups and sacrificed 14 days after study initiation. In all treatment groups, animals displayed normal behavior, with an average weight loss of less than 3%. A shorter 10-day study in animals treated with or without the Tween80 vehicle (n = 5), showed no effect on behavior or weight loss (data not shown). Lungs were removed and immediately frozen on dry ice and stored in RNA-later. Study protocol was approved by the local ethics committee (Malmö/Lund, Sweden, M103-14), with methods performed in accordance with relevant guidelines and regulations. Receptor binding and functionality profile of receptor antagonists EXT5 and EXT9 (AnaMar AB, Lund, Sweden) presents slightly separated receptor binding and functionality profiles as previously stated 4  , supplemented with 1% glutamine, 1% penicillin-streptomycin and 10% fetal clone serum (FCIII, Thermo Scientific, Waltham, MA, U.S.) at 37 °C, 10% CO 2 . Cells were seeded 5 000 cells/well in 96-well culture plates. After 6 h incubation, the cells were pre-treated 1 h with 5HT 2B receptor antagonists EXT5, EXT9 and RS 127445 (Tocris, Bristol, UK) (1 µM, 10 µM) (dissolved in DMSO) in DMEM medium with 1% serum. Cells were then treated with 5-HT (5-hydroxytryptamine hydrochloride, Sigma-Aldrich) (10 µM) in combination with 5-HT 2B receptor antagonists in 0.4% serum for 24 h. Cells were sub sequentially incubated with bromodeoxyuridine (BrdU) for additional 22 h ± 1 h. BrdU incorporation was measured using BrdU cell proliferation ELISA kit (ab126556, Abcam, Cambridge, U.K.), according to manufacturer's instructions. In a similar methodological manner, cell amount of HFL-1 cells was quantified after 48 h incubation with 5-HT (10 µM) and 5-HT 2B receptor antagonist (10 µM) treatment in 0.4% serum, using no pre-treatment with receptor antagonists. Cells were fixed with glutaraldehyde 1% and stained with crystal violet 0.1% for 30 min 34 . After repeated washing steps in ddH 2 O, cells were treated overnight with 1% Triton-X100 and absorbance was quantified at 595 nm.
Cell cycle assay. HFL-1 cells were seeded 400 000 cells in T25 cell culture flasks in supplemented DMEM with 10% FCIII. Cells incubated overnight at 37 °C, 10% CO 2 . Cells were pre-incubated with receptor antagonists (10 µM) in DMEM with 1% FCIII. The commercially available compound RS 127445 (5-HT 2B receptor antagonist) (Tocris, Bristol, UK) was used as a reference. After 1 h, cells were treated with 5-HT (10 µM) in combination with or without antagonists (10 µM). After 48 h incubation, cells were collected with trypsinization, counted and fixed for 30 min in ice cold 70% ethanol, at +4 °C. Cells were resuspended in D-PBS with 1% BSA and incubated with nuclear stain 7AAD (25 µg/ml, Sigma-Aldrich, A9400) and RNAseA (8 µg/ml, DNAse free, EN0531, Thermo Scientific) for minimum 10 min. Cells were acquired using flow cytometry in accuri C6 (BD Bioscience, San Jose, CA, U.S.) and analyzed with FlowJo LLC 10.3. Samples were run with slow flow rate with 20 000 events recorded per sample, with exclusion of doublets and debris by standard methodology. Linear format of FL3 were applied for detection of fluorescent emission per cell. Gating were set on 7AAD-peaks representing the standard G0/G1 and S, G2/M cell cycle phases. Data are presented as total cell count in S/G2/M phase in relation to all gated cells, shown in percent.
Statistical analysis. Detection of differentially expressed genes was done by using significance analysis of microarrays (SAM) according to the instructions in the software manual (Tmev software (MeV v. 4.2)). The different treatments were all compared to control samples, and probes that were significant after correction for multiple testing were used to create Venn diagram and extract common patterns between the different treatments. Analysis was performed in R software (v. 3.3.1). With rt-qPCR, one-way ANOVA was used to analyze relative gene expression levels in between treatment groups. For in vitro results, one sample t-test was applied as commonly recommended when the numbers in groups are small, as a non-parametric tests based on ranks seldom demonstrate any differences 35 . Statistical software program Graph Pad Prism 7 (La Jolla, CA), were used for statistical calculations on results obtained in vitro. P-values of *p < 0.05, **p < 0.01 and ***p < 0.001 were considered as statistical significant.
Data availability. The gene expression data that support the findings of this study are available from GEO data repository (NCBI) with further details described in Methods section. Remaining datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.