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Increased Expression of Heparan Sulfate 6-O-Sulfotransferase-2 Promotes Collagen Production in Cardiac Myofibroblasts
Kotaro KasaiYuma HoriiTakanori HironakaKyosuke MaeTomoyuki UenoAkiomi NagasakaMichio Nakaya
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2021 Volume 4 Issue 3 Pages 85-91

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Abstract

Fibrosis is defined as the excessive accumulation of extracellular matrix (ECM) proteins. These excessive ECM proteins are produced by myofibroblasts, which are differentiated mainly from resident fibroblasts in response to tissue injury. In addition to the ECM proteins, the amounts of heparan sulfate, one of the sugar chains, and the proteoglycans attached with heparan sulfate chains are reported to be increased in the fibrotic tissues. However, the contribution of heparan sulfate and heparan sulfate proteoglycans to the development of fibrosis remains unclear. In this study, we found that heparan sulfate 6-O-sulfotransferase-2 (Hs6st2), a type of heparan sulfate transferase, is remarkably induced during fibrosis in the heart, liver, and kidney of mice. We also demonstrated that Hs6st2 was specifically expressed in myofibroblasts of mice with cardiac and liver fibrosis. Hs6st2 knockdown in cardiac myofibroblasts reduced the mRNA expression of fibrosis-related factors, such as Collagen1a1. In summary, this study revealed that Hs6st2 is specifically expressed in myofibroblasts in fibrotic tissues, promotes fibrosis, and can be a good target for the treatment for fibrosis.

INTRODUCTION

Fibrosis is a pathological process common to a variety of tissues and diseases. It is defined as the excessive accumulation of the extracellular matrix (ECM), such as collagen and fibronectin, in the inflamed or damaged tissue, which can give rise to organ dysfunction.1,2) Although fibrosis is becoming increasingly recognized as a main cause of mortality in many diseases, there are few treatment strategies that can arrest or reverse fibrosis.1,2)

Myofibroblasts are the primary cells that produce and secrete greater levels of ECM proteins during fibrosis.3) In inflamed or damaged tissue, resident fibroblasts in the tissue mainly differentiate into myofibroblasts due to the stimulation of cytokines represented by transforming growth factor beta (TGF-β), secreted by macrophages.4,5) Although, to date, much effort has been paid to elucidate the molecular mechanisms that regulates the overproduction of ECM proteins, the understanding of the mechanisms is still limited.

Heparan sulfate proteoglycans are glycoconjugates in which sulfated sugar chains called heparan sulfate are attached to the core proteins.6,7) The heparan sulfate chain has a linear backbone composed of repeating disaccharides of glucuronic acid and N-acetylglucosamine.6,7) Heparan sulfate proteoglycans are involved in a variety of signaling pathways, such as growth factors and morphogenetic factors, which act as modulators that regulate the association of ligand molecules and receptors, and the storage and release of ligand molecules.7,8) In the fibrotic tissues, the amount of heparan sulfate9) and the expression levels of some heparan sulfate proteoglycans, such as Syndecan-1,1012) Glypican-3,13) and Perlecan,14) were reported to be increased. Syndecan-1 was also demonstrated to promote fibrosis in various tissues.1012) These reports suggested the physiological significance of heparan sulfate modification in fibrosis.

Heparan-sulfate 6-O-sulfotransferase (HS6ST) family, one of the heparan sulfate synthases, is an enzyme that catalyzes the sulfation of N-sulfoglucosamine at position 6.15,16) HS6ST family proteins are composed of three subtypes, HS6ST1, HS6ST2, and HS6ST3.17) A recent study has shown that the elevated expression of HS6ST1 is closely associated with the condition in the lungs of patients with idiopathic pulmonary fibrosis.18) However, the involvement of HS6ST2 and HS6ST3 in fibrosis has not been clarified. In this study, we explored the biological function of HS6ST2 in fibrosis.

MATERIALS AND METHODS

Mice

C57BL/6J mice were purchased from Japan SLC Inc. (Shizuoka, Japan). All mice studies were approved by the Animal Care and Use Committee of Kyushu University and followed the relevant national and international guidelines in the ‘Act on Welfare and Management of Animals’ (Ministry of Environment of Japan).

Myocardial Infarction (MI) Model

Male mice (8–10 weeks old) were anesthetized and subjected to permanent occlusion of the left coronary artery. For comparison, mice were subjected to the same procedure without the occlusion and these mice was defined as ‘sham’ group.

Non-Alcoholic Steatohepatitis (NASH) Model

Male mice (6 weeks old) were fed a choline-deficient, L-amino acid-defined high-fat diet with 60 kcal% fat and 0.1% methionine (CDAHFD) (Research Diets, NB, USA) for 10 weeks. For comparison, mice that were fed a normal diet (Research Diets) for 10 weeks were analyzed.

CCl4-Induced Liver Fibrosis Model

Male mice (8–10 weeks old) were intraperitoneally administered CCl4 [1 μL/g (Sigma-Aldrich, MO, USA), dissolved in corn oil (Sigma-Aldrich)], twice weekly for 4 weeks. For comparison, mice were administered vehicle (corn oil) and these mice was defined as ‘sham’ group. In addition, we prepared mouse group bred for more 4 weeks after the final CCl4 administration (termed as ‘Recover’ group).

Unilateral Ureteral Obstruction (UUO) Model

Anesthetized female mice (8–10 weeks old) were subjected to permanent occlusion of the left ureter. For comparison, mice that received the same procedure without the occlusion were analyzed. Ten days after the UUO operation, the kidneys of the mice were collected.

Real-Time RT-PCR Analysis

Total RNA from the tissues was collected by Isogen (Nippon gene, Tokyo, Japan) and purified using RNeasy Plus Mini Kit (QIAGEN, NRW, Germany). Total RNA from myofibroblasts was isolated using RNeasy Plus Mini Kit (QIAGEN). cDNA was synthesized using High-capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, MA, USA). Real-time RT-PCR was performed using Luna Universal qPCR Master Mix (New England BioLabs, MA, USA) on a StepOnePlus Real-Time PCR system (Applied Biosystems, CA, USA). Real-time RT-PCR probes were purchased from Applied Biosystems or Sigma-Aldrich and are described below.

Mouse Gapdh (Forward: 5′-CGTCCCGTAGACAAAATGGTGA-3′, Reverse: 5′-CCACTTTGCCACTGCAAATGG-3′, Probe: 5′-FAM-CCAATACGGCCAAATCCGTTCACACCGA -TAMRA-3′),

Mouse Hs6st1 (Forward: 5′-TGCGTTCGCCCAGAAAGTTC-3′, Reverse: 5′-GTCGCCATTCACTCAGGTAGC-3′, Probe: 5′-FAM-ATCACCCTGCTGCGAGACCCCGTAT-TAMRA-3′),

Mouse Hs6st2 (Forward: 5′-GCCAACAACCGCCAAGTTC-3′, Reverse: 5′-AACGCCATGTGCTTCAGATTG-3′, Probe: 5′-FAM-TGACCTGACTCTAGTGGGATGCTACAACCT-TAMRA-3′),

Mouse Hs6st3 (Forward: 5′-CCACAGCCACACCAGGAATTT-3′, Reverse: 5′-GGACATGCTTCCATTCGCTCA-3′, Probe: 5′-FAM-TGACACTGGGTCCCGCAACATCGTGA-3′),

Mouse Cd68 (Forward: 5′-CCGCTTATAGCCCAAGGAACA-3′, Reverse: 5′-TTCTGTGGCTGTAGGTGTCATC-3′, Probe: 5′-FAM-AAAGGCCGTTACTCTCCTGCCATCCT-TAMRA-3′),

Mouse Acta2 (Forward: 5′-CACCATGAAGATCAAGATCATTGCC-3′, Reverse: 5′-GGTAGACAGCGAAGCCAGGA-3′, Probe: 5′-FAM-AGCCACCGATCCAGACAGAGTACTTGCG-TAMRA-3′),

Mouse Col1a1 (Forward: 5′-CCCAAAGGTTCTCCTGGTGAAG-3′, Reverse: 5′-CGGTTTTGCCATCAGGACCA-3′, Probe: 5′-FAM-TGGTGCCAAGGGTCTCACTGGCAGTC-TAMRA-3′),

Mouse Col8a1 (Forward: 5′-CGAGAGGGGAAAAAGGACCCA-3′, Reverse: 5′-TGGTCCTACAACTCCACCTTCTC-3′, Probe: 5′-FAM-AGGTGCTATTGGTTTCCCTGGACCCA-TAMRA-3′),

Mouse Lox (Forward: 5′-CAAGCTGGTTTCTCGCCGTC-3′, Reverse: 5′-TGTAGGGGTCGTCGCCCA-3′, Probe: 5′-FAM-ATCGCCACAGCCTCCGCAGCTCAG-TAMRA-3′),

Mouse Il6 (Forward: 5′- TGGAAATGAGAAAAGAGTTGTGC-3′, Reverse: 5′- TCCAGTTTGGTAGCATCCATCA-3′, Probe: 5′-FAM- TTCTGCAAGTGCATCATCGTTGTTCATACA-3′),

Mouse Cyp7a1 Mm00484150_m1

Relative gene expression was determined using the comparative Ct method. Gapdh mRNA was amplified as an internal control. In the absolute quantification of Hs6st1 or Hs6st2 mRNA, pMD20‐Hs6st1 (NM_015818.2, coding sequence of 282–853 bp) and pMD20‐Hs6st2 (NM_001290467.1, coding sequence of 934–1500 bp) were used to create standard curves. The copy numbers of Hs6st1 and Hs6st2 in 1 ng RNA were determined from the standard curves.

Isolation of Cardiac Myofibroblasts by Magnetic-Activated Cell Sorting (MACS)

The hearts of the mice were collected three days after the MI operation and digested using 0.1% Collagenase type II (Worthington, NJ, USA), 0.01% Elastase (Worthington), and 2.5 mg/mL DNase I (Sigma Aldrich) in phosphate-buffered saline (PBS) (Nacalai Tesque, Kyoto, Japan) at 37°C for 1 h under shaking. The cells were collected and treated with red blood lysis buffer (Roche, BL, Swiss) and plated in Dulbecco’s modified eagle’s medium (Nacalai Tesque), containing 10% fetal bovine serum (Thermo Fisher Scientific) and 1% penicillin-streptomycin (Nacalai Tesque) for 6 h. Then, the culture medium was exchanged with fresh medium, and the cells were allowed to grow overnight at 37°C. After overnight culture, the cells were collected by accutase (Nacalai Tesque) treatment, washed using MACS buffer (0.5% bovine serum albumin [BSA]/2 mM EDTA (DOJINDO, Kumamoto, Japan)/PBS), and incubated with CD45 microbeads on ice for 20 min (Miltenyi Biotec, NRW, Germany). The CD45-negative cells were isolated using MACS column and were used as cardiac myofibroblasts.

Isolation of Hepatocytes, Kupffer Cells, and Hepatic Stellate Cells (HSCs) by MACS

After the 4-week-administration of CCl4, the livers of mice were perfused with wash buffer [25 mM HEPES (DOJINDO)/0.5 mM EDTA/HBSS (Nacalai Tesque)] at 37°C through the portal vein and subsequently with digestion buffer [0.1% Collagenase A (Roche)/1.5 mM HEPES/HBSS]. Next, the livers were collected and digested in digestion buffer at 37°C for 30 min. The cells were subjected to centrifugation [50 × g, 1 min] twice, and divided into the precipitated cell fraction and the supernatant cell fraction. The precipitated cell fraction was collected and plated on a 0.1% (w/v) gelatin-coated dish for 6 h. The adherent cells were collected by accutase treatment, and washed using MACS buffer, followed by incubation with CD45 microbeads on ice for 20 min. The CD45-negative cells were isolated using MACS column and were then used as hepatocytes. The supernatant cell fraction was separated using F4/80 microbeads (Miltenyi Biotec) and the F4/80-positive cells were collected as Kupffer cells. The F4/80-negative cells were incubated with CD45 microbeads and subsequently subjected to MACS separation. CD45-negative cells were collected as HSCs.

In situ Hybridization

Seven days after the MI operation, the hearts of the mice were fixed in 4% paraformaldehyde (Nacalai Tesque) overnight, frozen with Tissue-Tek O.C.T. Compound (Sakura, Tokyo, Japan), and cut into 6-μm sections. The sections were subjected to RNAScope® Multiplex Fluorescent v2 Assay kit (Advanced Cell Diagnostics, CA, USA) using RNAScope® probe specific for Hs6st2, according to the manufacturer’s instructions. Subsequently, the sections were incubated with blocking buffer (10% BSA/PBS) and incubated with rabbit anti-αSMA antibody (1:200; ab5694, abcam, Cambs, England) or rat anti-CD45 antibody (1:200; 103119, Biolegend, CA, USA), diluted in blocking buffer, overnight at 4°C. After PBS wash, the sections were incubated with Alexa488-conjugated donkey anti-rabbit IgG antibody (1:200; A-21206, Invitrogen, CA, USA) or Alexa488-conjugated donkey anti-rat IgG antibody (1:200; A-21208, Invitrogen), diluted in blocking buffer, for 1 h at 20°C, washed using PBS, and incubated with 4′,6-diamidino-2-phenylindole (1:1000, DOJINDO), diluted in PBS, for 5 min. After PBS wash, the sections were sealed and observed using confocal microscope (LSM700).

Knockdown Analysis

Myofibroblasts were treated with small interfering RNA (siRNA) (50 pmol, mouse Hs6st2: s78481 control: 4390844, life technologies, CA, USA) using LipofectamineTM RNAiMAX (Invitrogen). Total RNA was isolated 48 h later.

Statistical Analysis

The statistical analyses were performed using PRISM 5 (GraphPad Prism software, CA, USA). The results were described as the means ± standard error of mean. The results were statistically analyzed using the unpaired Student’s t-test or one-way ANOVA, followed by Tukey’s multiple comparison tests.

RESULTS

Hs6st2 Expression was Remarkably Increased in Fibrotic Heart, Liver, and Kidney

To examine the contribution of Hs6st family members to cardiac fibrosis, we first examined the mRNA expression levels of Hs6st family members, Hs6st1, Hs6st2, and Hs6st3 in the hearts of mice after MI treatment, which is known to induce cardiac cell death, resulting in the inflammation and fibrosis in the heart. We found that the mRNA level of Hs6st1 was expressed in the healthy mouse hearts (Fig. 1A), and the expression level of Hs6st1 was significantly increased in the infarct region but not in non-infarct region of the hearts of mice, 3 or 7 d after MI (Fig. 1B). In contrast, Hs6st2 was hardly expressed in healthy mouse hearts (Fig. 1A). However, its expression was greatly increased in the infarct region, but not in non-infarct region of the hearts of mice 3, 7, or 28 d after MI (Fig. 1B). The increased expression of Col8a1 in the infarct region of the hearts of mice 3, 7, or 28 d after MI confirmed that fibrotic responses occurred in the region (Fig. 1B). Hs6st3 was not expressed in healthy or infarcted hearts of mice (data not shown). These results suggested that the increase in the mRNA expression of Hs6st1 and Hs6st2 was linked to cardiac fibrosis.

Fig. 1

The mRNA Expression Levels of Hs6st2 were Significantly Increased in the Fibrotic Heart, Liver, and Kidney of Mice

(A) Absolute quantification of the gene expression of Hs6st1 and Hs6st2 in healthy mouse hearts by real-time RT-PCR (n = 3 each). (B) The mRNA expression levels of Hs6st1, Hs6st2, and Col8al in mouse hearts 0, 3, 7, and 28 d after MI operation. ‘Sham’ means sham-operated mouse hearts. ‘Infarct’ or ‘Non-infarct’ means infarct region or non-infarct region of mouse hearts after MI. ***P < 0.001, *P < 0.05 Infarct vs Sham. ###P < 0.001, #P < 0.05 Infarct vs Non-infarct. n = 3–6. (C) Absolute quantification of the gene expression of Hs6st1 and Hs6st2 in livers of mice fed with a standard diet by real-time RT-PCR (n = 7 each). (D) The mRNA expression levels of Hs6st1, Hs6st2, and Col8al in the livers of mice fed with a standard diet (SD) or mice with CDAHFD-induced NASH (n = 4–7). (E) The mRNA expression levels of Hs6st2 and Col8al in livers of mice after 4 weeks of administration of corn oil, used as vehicle (Veh) or CCl4, or livers CCl4-administered mice after 4 weeks recovery (Rec) (n = 5–6). (F) The mRNA expression levels of Hs6st1, Hs6st2, and Col8al in the kidneys of mice after sham or UUO operation (n = 5). The mRNA levels were normalized to those of Gapdh (B, D–F) and shown as fold changes relative to day 0 (B), SD (D), Veh (E) or Sham (F). Data are presented as the mean ± SEM. For (A–F), ***P < 0.001, **P < 0.01, n.s.: not significant.

We then examined whether the increase in the mRNA expression levels of Hs6st1 and Hs6st2 was also observed in hepatic fibrosis. Male mice were fed a CDAHFD for 10 weeks to induce a mouse model of NASH with fibrosis. Results from the real-time RT-PCR analysis revealed that Hs6st1 was expressed in normal mouse liver fed with standard diet (Fig. 1C), and its expression level was not increased in NASH mouse liver (Fig. 1D). In contrast, Hs6st2 was not expressed in normal mouse liver (Fig. 1C), and the expression levels of Hs6st2 and Col8a1 were strikingly increased in NASH mouse liver (Fig. 1D). Hs6st3 was not expressed in healthy or NASH livers of mice (data not shown).

In addition, the mRNA expression of Hs6st2 and Col8a1 were greatly increased in the fibrotic liver of mice after 4 weeks of administration of CCl4, which is known to induce liver injury (Fig. 1E). The liver is a highly regenerative organ. It was observed that the livers of mice that were bred for 4 more weeks after the final CCl4 administration were quite similar to the normal mouse liver. Interestingly, 4 weeks after withdrawal of CCl4 administration, Hs6st2 and Col8a1 mRNA expressions were significantly reduced, reaching to the level in the vehicle group (Fig. 1E). These findings suggested that the expression level of Hs6st2 was correlated with the extent of hepatic fibrosis.

We further examined the increase in the expression of Hs6st1 and Hs6st2 in fibrotic kidney. For the experiment, we used an UUO model, which is a widely used model of renal fibrosis. As observed in case of the liver, the expression level of Hs6st1 was not altered in fibrotic kidney, while that of Hs6st2 and Col8a1 were greatly increased (Fig. 1F). Hs6st3 was not expressed in healthy or UUO kidneys of mice (data not shown). Taken together, these results indicated that Hs6st2 mRNA expression was increased in the fibrotic heart, liver, and kidney of mice.

Hs6st2 was Specifically Expressed in the Myofibroblasts of Fibrotic Heart of Mouse

We then examined the cells that express Hs6st2 in the fibrotic heart of mice by in situ hybridization. In the section of mice hearts after sham operation, the signals for Hs6st2 mRNA were rarely detected (Fig. 2A). In contrast, the signals were abundant in infarct area of the mice hearts, 7 d after MI. Close observation of the sections also revealed that the signals for Hs6st2 mRNA were exclusively found in the interstitium of the infarcted mice hearts. The main population of the cells that reside in the interstitium of the infarcted mice hearts are myofibroblasts or leukocytes, though the number of myofibroblasts is much larger than that of leukocytes. Therefore, we examined whether these cells express Hs6st2. In situ hybridization to detect Hs6st2 mRNA, and the immunostaining for alpha smooth muscle actin (αSMA), a marker of myofibroblasts, were performed on the same heart sections. We found that almost all Hs6st2 signals were detected in the myofibroblasts (Fig. 2B). However, the signals were not found in the cells that express CD45, a marker of leukocytes (Fig. 2C). αSMA is the most commonly used marker molecule for myofibroblasts, but it is well known that αSMA is more highly expressed in smooth muscle than in myofibroblasts. Interestingly, we found that Hs6st2 was not expressed in the vascular smooth muscle cells present around the vessel, suggesting that Hs6st2 may be a better marker molecule than αSMA, at least in the fibrotic hearts of mice (Fig. 2D).

Fig. 2

Hs6st2 is Specifically Expressed in Cardiac Myofibroblast and Hepatic Stellate Cells

(A–D) Representative in situ hybridization images detecting Hs6st2 mRNA in mice left ventricle of sham (A) or 7 d after MI operation (B–D). The sections were co-stained with an anti-αSMA (green) (B, D) or anti-CD45 (green) (C) antibody. Yellow arrowheads indicate the signals for Hs6st2 (red) detected in myofibroblasts that express αSMA. The percentage of the Hs6st2-positive cells (n = 150) that are also αSMA-positive were quantified and shown in a graph. White arrowheads indicate leukocytes. Blue arrowheads indicate vascular smooth muscle cells. BV in the images of (D) indicates the blood vessels. Scale bars in (A–D) = 20 µm. (E) The mRNA expression levels of Cyp7a1, Cd68, Acta2 and Hs6st2 in hepatocytes (HC), Kupffer cells (KC) and hepatic stellate cells (HSC) isolated from a CCl4-induced fibrotic mouse liver (n = 1). mRNA levels were normalized to those of Gapdh and shown as fold changes relative to HC.

We further examined whether Hs6st2 was also expressed in myofibroblasts of fibrotic livers. Since it was technically difficult to perform in situ hybridization using the livers of NASH mice and CCl4-treated mice, we fractionated the cell population of hepatocytes, Kupffer cells (liver macrophages), and hepatic stellate cells (liver myofibroblasts) by centrifugation and MACS. We confirmed the purity of hepatocytes, Kupffer cells, and myofibroblasts by measuring the mRNA expression levels of Cyp7a1 (a hepatocyte marker), Cd68 (a macrophage marker), and Acta2 (encodes αSMA) (Fig. 2E). Results from the real-time RT-PCR analysis revealed that Hs6st2 expression was only detected in the myofibroblast population, indicating that Hs6st2 was also expressed in myofibroblasts of fibrotic livers (Fig. 2E).

Hs6st2 Knockdown Attenuated the Expression of Fibrosis-Related Genes in Cardiac Myofibroblasts

We finally investigated the role of Hs6st2 in cardiac myofibroblasts. Comparison of Hs6st1 and Hs6st2 mRNA expression levels in the cardiac myofibroblasts, using absolute quantification methods, revealed that the mRNA expression level of Hs6st1 was markedly lower than that of Hs6st2 (Fig. 3A). Since the main function of cardiac myofibroblasts is the production of extracellular matrix proteins, we examined the effect of Hs6st2 on their production. For this purpose, we treated cardiac myofibroblasts with siRNA targeting Hs6st2 and measured the mRNA expression levels of Col1a1, Col8a1, and Lox by real-time RT-PCR analysis. The analysis demonstrated that their expression levels were significantly decreased by Hs6st2 knockdown (Fig. 3B). On the other hand, the expression level of Il6, an inflammatory gene, was not changed by Hs6st2 knockdown (Fig. 3C). These results implied that Hs6st2 enhances the fibrosis by increasing the expression of collagen genes in cardiac myofibroblasts.

Fig. 3

Hs6st2 Knockdown Decreases the Expression Levels of Fibrosis-Related Genes in Cardiac Myofibroblasts

(A) Absolute quantification of the gene expression of Hs6st1 and Hs6st2 in cardiac myofibroblasts by real-time RT-PCR (n = 5 each). (B) The mRNA expression levels of Hs6st2, Col1a1, Col8a1, and Lox in cardiac myofibroblasts after treatment of siRNA targeting Hs6st2 or siCtrl (n = 5 each). (C) The mRNA expression level of Il6 in cardiac myofibroblasts after treatment of siRNA targeting Hs6st2 or siCtrl (n = 5 each). The mRNA levels were normalized to those of Gapdh and shown as fold changes relative to siCtrl. Data are presented as the mean ± SEM. ***P < 0.001, **P < 0.01, n.s.: not significant.

DISCUSSION

We demonstrated that Hs6st2 expression was markedly increased during fibrosis in the heart, liver, and kidney of mice. Hs6st2 was hardly expressed in the normal mouse heart but was specifically expressed in myofibroblasts of the fibrotic hearts after MI. Hs6st2 expressed in the myofibroblasts was found to promote the production of extracellular matrix proteins, such as collagen.

Cardiac myofibroblasts are reported to differentiate primarily from resident fibroblasts present in the normal heart.5) Since Hs6st2 is not expressed in the resident fibroblasts in the normal mouse heart, the mRNA expression of Hs6st2 is considered to increase with the differentiation from the resident fibroblasts into myofibroblasts. One of the important signals for myofibroblast differentiation is TGF-β signaling.4) Therefore, Hs6st2 expression may be regulated by TGF-β signaling. In contrast to the expression of Hs6st2, the expression of Hs6st1, a molecule belonging to the same Hs6st family, showed little change in during fibrosis (Fig. 1). Therefore, in cardiac fibroblasts/myofibroblasts, the expression levels of Hs6st1 and Hs6st2 were considered to be controlled by different mechanisms. Interestingly, it has been reported that unlike in cardiac myofibroblasts, the expression level of Hs6st1 increases in lung myofibroblasts during pulmonary fibrosis, whereas the expression level of Hs6st2 does not change in the cells.18) At present, it is not clear why the types of Hs6st that increase in expression with fibrosis in the heart and lung are different. However, since both Hs6st1 and Hs6st2 have the function of adding sulfate group to N-sulfoglucosamine,1517) it is considered that they may add sulfate group to N-sulfoglucosamine of the same proteins that promote collagen production in the fibrotic heart and lung. One of the candidate proteins is Syndecan-1, a heparan sulfate proteoglycan that is modulated by the HS6ST family.19) Syndecan-1 is known to increase its expression in tissues with fibrosis.1012) Therefore, it may be interesting to detect the changes in the glycosylation of Syndecan-1 during the differentiation into myofibroblasts.

It has been known that Hs6st2 promotes the signal transduction mediated by growth factors, such as epidermal growth factor (EGF) and fibroblast growth factor (FGF).20,21) In fact, in the adipocytes of Hs6st2 KO mice, in which increased lipid accumulation in liver was observed, FGF19 and 21 downstream signaling was attenuated.22) The myofibroblast differentiation was promoted by various cytokines, such as TGF-β, platelet-derived growth factor (PDGF) and FGF.23,24) Therefore, HS6ST2 may contribute to the myofibroblast differentiation by modulating these cytokine-elicited pathways. In this context, it would be interesting to examine whether the FGF downstream signaling was decreased in Hs6st2 knock downed myofibroblasts.

In the heart, Hs6st2 is expressed specifically in myofibroblast and it promotes the expression of fibrotic factors, such as collagen. The molecular mechanisms that regulate fibrosis in each tissue are still obscure, and the development of the effective drug for fibrosis is desired. Since Hs6st2 is rarely expressed in the normal heart, liver, and kidney, it may be a good therapeutic target for fibrotic diseases with low risk of side effects.

Acknowledgments

We would like to thank Dr. H. Kurose for his kind support throughout this research. This study was supported by grants from Grants-in-Aid for Scientific Research (KAKENHI) [to M.N. (JP22590083, JP25670120, JP20H03383) and A.N. (JP19K07122)]; The Takeda Science Foundation, The Mochida Memorial Foundation for Medical and Pharmaceutical Research, 2018 Bristol-Myers Squibb KK Research Grants, The Salt Science Research Foundation, The Koyanagi Foundation, The Nakatomi Foundation, The Suzuken Memorial Foundation (to M.N.); from Japan Agency for Medical Research and Development (AMED)(JP20gm5810030 to M.N.); from Grant-in-Aid for JSPS Research Fellow (JP19J20083 to Y.H., JP21J11273 to T.H.) ; from Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under Grant Number JP19am0101091. We appreciate for the technical supports from the Research Support Center, Graduate School of Medical Sciences, Kyushu University and from Medical Institute of Bioregulation, Kyushu University.

Conflict of interest

The authors declare no conflict of interest.

REFERENCES
 
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