FGF-2, IL-1b and TGF-b regulate fibroblast expression of S100A8 Farid Rahimi, Kenneth Hsu, Yasumi Endoh and Carolyn L Geczy Inflammatory Diseases Research Unit, School of Medical Sciences, University of New South Wales, Sydney, Australia Keywords FGF-2; fibroblasts; interleukin-1b; S100A8 gene; TGF-b Correspondence C Geczy, Inflammatory Diseases Research Unit, School of Medical Sciences, The University of New South Wales, Sydney, NSW 2052, Australia Fax: + 61 293851389 Tel: + 61 293851599 E-mail: c.geczy@unsw.edu.au Website: http://www.med.unsw.edu.au/ (Received March 2005, revised 28 March 2005, accepted April 2005) doi:10.1111/j.1742-4658.2005.04703.x Growth factors, including fibroblast growth factor-2 (FGF-2) and transforming growth factor-b (TGF-b) regulate fibroblast function, differentiation and proliferation S100A8 and S100A9 are members of the S100 family of Ca2+binding proteins and are now accepted as markers of inflammation They are expressed by keratinocytes and inflammatory cells in human ⁄ murine wounds and by appropriately activated macrophages, endothelial cells, epithelial cells and keratinocytes in vitro In this study, regulation and expression of S100A8 and S100A9 were examined in fibroblasts Endotoxin (LPS), interferon c (IFNc), tumour-necrosis factor (TNF) and TGF-b did not induce the S100A8 gene in murine fibroblasts whereas FGF-2 induced mRNA maximally after 12 h The FGF-2 response was strongly enhanced and prolonged by heparin Interleukin-1b (IL-1b) alone, or in synergy with FGF-2 ⁄ heparin strongly induced the gene in 3T3 fibroblasts S100A9 mRNA was not induced under any condition Induction of S100A8 in the absence of S100A9 was confirmed in primary fibroblasts S100A8 mRNA induction by FGF-2 and IL-1b was partially dependent on the mitogen-activated-proteinkinase pathway and dependent on new protein synthesis FGF-2-responsive elements were distinct from the IL-1b-responsive elements in the S100A8 gene promoter FGF-2- ⁄ heparin-induced, but not IL-1b-induced responses were significantly suppressed by TGF-b, possibly mediated by decreased mRNA stability S100A8 in activated fibroblasts was mainly intracytoplasmic Rat dermal wounds contained numerous S100A8-positive fibroblastlike cells and days post injury; numbers declined by days Up-regulation of S100A8 by FGF-2 ⁄ IL-1b, down-regulation by TGF-b, and its timedependent expression in wound fibroblasts suggest a role in fibroblast differentiation at sites of inflammation and repair Fibroblasts are heterogeneous stromal resident cells that participate in wound healing, fibrosis ⁄ scarring and immune ⁄ inflammatory processes [1,2] by contributing to leukocyte recruitment ⁄ accumulation, angiogenesis, matrix metabolism, and protection against oxidative damage [3,4] Numerous factors including extracellular matrix (ECM) components, some cytokines, prostaglandins, reactive oxygen species (ROS), and growth factors [5] modulate fibroblast function Abbreviations ActD, actinomycin D; BCS, bovine calf serum; BM, bone marrow; BMF, bone-marrow-derived fibroblast-like cells; C ⁄ EBP, CCAAT ⁄ enhancer binding protein; CM, culture medium; DAPI, 4,6-diamidino-2-phenylindole; DMEM, Dulbecco’s modified Eagle’s medium; DPBS, Dulbecco’s phosphate-buffered saline; DTT, dithiothreitol; ECM, extracellular matrix; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; FGF, fibroblast growth factor; Hepes, N-2-hydroxyethylpiperazine-N ¢-2-ethanesulfonic acid; HPRT, hypoxanthine phosphoribosyl-transferase; HRP, horseradish-peroxidase; IFNc, interferon c; IL-1b, interleukin-1b; JNK, c-Jun N-terminal kinase; LDL, low-density lipoprotein; LPS, endotoxin; mS100A8, murine S100A8; MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; mOxS100A8, HOCl-oxidized murine S100A8; PKC, protein kinase C; ROS, reactive oxygen species; SPF, splenic ‘primary’ fibroblast-like cells; TGF-b, transforming growth factor-b; TNF, tumor-necrosis factor FEBS Journal 272 (2005) 2811–2827 ª 2005 FEBS 2811 Expression and regulation of S100A8 in fibroblasts S100 Ca2+-binding proteins may have evolved from a calmodulin ancestor [6] and are implicated in numerous intra ⁄ extracellular processes and can act as Ca2+ sensors [7] Some (e.g S100A6 (calcyclin) [8], S100A4 [9] and S100A11 [10,11]) have been implicated in fibroblast growth and differentiation S100A12 acts as a pro-inflammatory ‘cytokine’ [12,13] and S100A12 mRNA is induced by interleukin-1a (IL-1a) and tumor necrosis factor (TNF) in bovine corneal fibroblasts [14] S100B is also expressed by fibroblasts [15] and may be involved in regulation of growth arrest and apoptosis [16], stimulation of cell proliferation [17] and protection against apoptosis [18] Other S100 proteins also have proliferative and anti-apoptotic effects [8,9,19] S100A8 and S100A9 (calgranulins A and B; MRP8 and MRP14) have been associated with leukocyte differentiation, inflammation and wound healing [20,21] They are proposed to be involved in reorganization of the keratin cytoskeleton and differentiation of keratinocytes and in antibacterial or antioxidant defense in the wounded or normal epidermis [20–22] Some functions are dependent on S100A8–S100A9 heterodimers, e.g arachidonic acid binding, antimicrobial defense and regulation of NADPH-oxidase activity (reviewed in [20]) S100A9 and the S100A82–S100A9 complex (calprotectin) stimulate IL-8 production by airway epithelial cells, hence potentially amplifying neutrophilic inflammation in chronic pulmonary disease [23] Murine S100A8 is a potent leukocyte chemoattractant [24] and intracellular S100A8 and S100A9 essentially regulate phagocyte migration by integrating the calcium and mitogen activated protein kinase (MAPK) transduction signals thereby controlling reorganization of the phagocyte microtubular system [25] Expression of S100A8 in the absence of S100A9 in activated murine macrophages [26,27] and keratinocytes [22], and presence of S100A8 without S100A9 in the low density lipoprotein (LDL) proteome [28] is strong evidence that S100A8 does not depend on S100A9 for structural stability [29] and strengthens the proposal for independent function We showed that S100A8 scavenges O2– and hypochlorite, suggesting a role in oxidative defense [22,30,31] The S100A8 gene is up-regulated by anti-inflammatory mediators [27], corticosteroids [32] and by oxidative stress [22] indicating a protective function Other S100 proteins are also implicated in cellular responses to oxidative stress In particular in keratinocytes, S100A2 oxidation and translocation were proposed as early markers of oxidative stress and were markedly attenuated in malignant keratinocytes, favoring a role in oxidant defense rather than in tumor proliferation [33] 2812 F Rahimi et al Here we show that factors important in wound healing regulate S100A8, but not S100A9, in fibroblasts Fibroblast growth factor-2 (FGF-2) and IL-1b strongly induced S100A8 via a MAPK-dependent pathway Responses to FGF-2 were amplified by heparin and there was strong synergy between FGF-2 and IL1b The protein was cytoplasmic TGF-b suppressed S100A8 induction by FGF-2 but not by IL-1b, suggesting important regulatory differences, and promoter analysis confirmed different enhancer elements regulating induction by IL-1b and FGF-2 In a rat incisional wound, immunohistochemical studies showed S100A8 expression in fibroblast- and macrophage-like cells, keratinocytes and neutrophils in the incision area We propose that S100A8 may be involved in pathways regulating fibroblast growth and differentiation, possibly by regulating intracellular redox, at sites of inflammation and ⁄ or repair ⁄ remodeling Results FGF-2 induces S100A8 mRNA in 3T3 fibroblasts Initially, induction of murine S100A8 (mS100A8) mRNA in 3T3 fibroblasts was variable, suggesting that, like microvascular endothelial cells [34], cell–cell contact may be important Fibroblast monolayers grown to various states of confluence, were stimulated with FGF-2 ± heparin and harvested after 24 h Unstimulated confluent (C, Fig 1) or subconfluent (not shown) 3T3 cells had little detectable S100A8 mRNA whereas the bone-marrow (BM) RNA was positive (BM, Fig 1) FGF-2 (1.5 nm) weakly induced the S100A8 gene and responses gradually increased from  21–51% of maximum in cells grown from  30–80% confluence; levels were maximal at confluence (Fig 1) Heparin modulates FGF-2-receptor binding and activity [35] and although it had no direct influence (Fig 6), S100A8 mRNA increased approximately two-fold in confluent 3T3 cells stimulated with FGF-2 and heparin compared to FGF-2 alone Potentiation was minimal in cells grown to  80% confluence (Fig 1) indicating that cell–cell contact is important for optimal induction of S100A8 mRNA expression in 3T3 fibroblasts Real-time RT-PCR confirmed that S100A9 was not induced in confluent cells stimulated with FGF-2 with ⁄ without heparin Kinetics of induction of S100A8 mRNA in 3T3 fibroblasts mS100A8 mRNA induction in FGF-2-heparin-activated fibroblasts was dose- and time-dependent Up to FEBS Journal 272 (2005) 2811–2827 ª 2005 FEBS F Rahimi et al Expression and regulation of S100A8 in fibroblasts A Fig S100A8 mRNA induction in fibroblasts depends on confluence 3T3 cells grown to approximately 30, 80 and 100% confluence were stimulated with FGF-2 (1.5 nM) in the presence (+) or absence (–) of heparin (1 ImL)1) for 24 h Total RNA was examined by Northern blotting using an S100A8 riboprobe and a rat 18S rRNA oligoprobe as indicated BM indicates murine bone-marrow RNA (positive control) and C, the negative control (unstimulated cells) The graph indicates percentage maximum of the normalized response Similar results observed in at least three experiments 0.15 nm FGF-2 with ImL)1 heparin did not induce detectable mRNA (Fig 2A), whereas 1.5 nm FGF-2 and heparin induced strong responses that were maximal with nm FGF-2 (Fig 2A); and 15 nm FGF-2 induced mS100A8 mRNA levels that were  80 and 60% of maximal expression, respectively (Fig 2A), suggesting production of a suppressor Heparin generated maximal responses at and 10 ImL)1 (Fig 2B) whereas higher amounts (50 and 100 ImL)1) reduced responses to  72 and 48% of maximum, respectively, in cells costimulated with 1.5 nm FGF-2 (Fig 2B) possibly due to soluble heparin-mediated inhibition of FGF-2-receptor binding [35] For subsequent experiments, 1.5 nm FGF-2 with ImL)1 heparin were used to stimulate confluent fibroblasts S100A8 mRNA induction in 3T3 fibroblasts activated with FGF-2 was evident after h and in the presence of heparin, mRNA levels increased in parallel up to 12 h when the response to FGF-2 was maximal and gradually declined over 36 h (Fig 2C) Potentiation by heparin was most apparent at 18 h, when mRNA levels were approximately double those with FGF-2 alone at 12 h In FGF-2-heparin-stimulated cells, S100A8 mRNA levels were maintained for up to 36 h and declined to 20% of maximum by 48 h (Fig 2C) FEBS Journal 272 (2005) 2811–2827 ª 2005 FEBS B C Fig Dose- and time-dependence of induction of S100A8 mRNA by FGF-2 and heparin in confluent 3T3 cells (A) Confluent 3T3 cells treated with FGF-2 (1.5 pM-15 nM) and heparin (1 ImL)1) for 24 h and total RNA analyzed by Northern blotting; percentage maximum response is given in the bar graph; C and BM indicate unstimulated fibroblasts’ and bone-marrow RNA, respectively (B) 3T3 cells treated with 1.5 nM FGF-2 were costimulated with increasing amounts of heparin (0.01–100 ImL)1) (C) Kinetics of induction of S100A8 mRNA 3T3 cells treated with FGF-2 (1.5 nM) or FGF-2 ⁄ heparin (1 ImL)1) were harvested at the times indicated and Northern analysis performed Similar results observed in three different experiments Because the mS100A8 gene in elicited macrophages and other cells [26,34] is induced by particular proinflammatory mediators, endotoxin (LPS, 250– 2813 Expression and regulation of S100A8 in fibroblasts 1000 ngỈmL)1), interferon c (IFNc, 500 mL)1), or TNF (30 ngỈmL)1) were tested No S100A8 mRNA was detected in 3T3 cells stimulated for 24 h although mRNA was substantially augmented with a combination of LPS, IFNc, and TNF and confluence influenced levels (not shown) Induction was maximal after 24 h and decreased to  47% of maximum by 48 h (not shown) IL-1b-stimulated 3T3 fibroblasts express S100A8 mRNA IL-1b is a strong inducer of the S100A8 gene in microvascular endothelial cells [34] IL-1b induced S100A8 mRNA in confluent 3T3 fibroblasts in a dose- and time-dependent manner As little as mL)1 was effective and responses were maximal with 10– 20 mL)1 (Fig 3A); 10 mL)1 IL-1b were used routinely mRNA was detected after h, gradually increased to 28% of maximum after 24 h, was maximal at 32 h and declined to  30% of maximum after 48–52 h (Fig 3B) S100A8 mRNA levels in 3T3 cells stimulated with FGF-2 ⁄ heparin almost tripled when costimulated with 0.1 mL)1 IL-1b and were augmented  7.5-fold with mL)1 Maximal up-regulation was with A F Rahimi et al Table S100A8 mRNA levels assayed in activated 3T3 cells by real-time RT-PCR 3T3 cells unstimulated or treated with IL-1b (10 mL)1), FGF-2 (1.5 nM) + heparin (1 ImL)1) or their combination for 24 h mS100A8, mS100A9, and HPRT mRNA levels were quantitated by real-time RT-PCR S100A8 expression was normalized to that of HPRT and expressed as fold-increase compared to medium control The data are means and standard errors of duplicate measurements S100A9 mRNA was not detected Treatment S100A8 induction S100A9 induction Medium control IL-1b FGF-2 + heparin FGF-2 + heparin + IL-1b 1.0 97.0 74.54 5907 0 0 ± ± ± ± 0.064 8.48 13.89 390.2 10 mL)1 IL-1b which generated a 14.2-fold increase compared to 10 or 20 mL)1 IL-1b alone, or FGF-2 and heparin without IL-1b (Fig 3C) To quantitate this more accurately, relative S100A8 mRNA levels were analyzed by real-time RT-PCR Table shows that IL-1b (10 or 20 mL)1) induced mRNA levels similar to those induced by FGF-2 and heparin The magnitude of synergy was more apparent, with  580fold more S100A8 mRNA in cells costimulated with FGF-2, heparin and IL-1b compared to those stimulated with FGF-2 and heparin B C Fig Effects of IL-1b on S100A8 mRNA induction (A) Northern analysis of mRNA from confluent 3T3 cells stimulated with the given doses of IL-1b for 24 h Results represent three experiments (B) 3T3 cells stimulated with 10 mL)1 IL-1b were harvested at the times indicated The line graph indicates normalized levels of S100A8 mRNA Similar results observed in two experiments (C) Northern blot analysis of confluent 3T3 cells stimulated for 24 h with FGF-2 (F, 1.5 nM) and heparin (H, ImL)1) in the presence of increasing doses of IL-1b; data representative of at least three different experiments (D) Confluent SPF stimulated with FGF-2 and heparin and IL-1b at the doses indicated The bar graph indicates normalized levels of S100A8 mRNA 2814 FEBS Journal 272 (2005) 2811–2827 ª 2005 FEBS D F Rahimi et al Induction of S100A8 mRNA in primary murine fibroblast-like cells Because fibroblasts are phenotypically and functionally heterogeneous and possess unique subpopulations even in the same tissue [36,37] and because 3T3 fibroblasts may not be representative of tissue fibroblasts, the S100A8 gene regulation was assessed in different fibroblast populations and RNA from splenic ‘primary’ fibroblast-like cells (SPF) and bone marrow-derived fibroblast-like cells (BMF) were analyzed Confluent SPF contained no detectable S100A8 mRNA by northern analysis (Fig 3D) and cells stimulated for 24 h with IL-1b (samples and 9), FGF-2 (sample 10), or FGF-2 and heparin (samples and 5) did not express the gene IL-1b and FGF-2 combined (samples and 7, Fig 3D) weakly induced mRNA ( 38–47% of maximum), responses at 24 h were maximal with nm FGF-2, ImL)1 heparin and 10 mL)1 IL-1b (sample 3) and half-maximal with mL)1 IL-1b and 1.5 nm FGF-2 (sample 2, Fig 3D) The same pattern was observed with BMF stimulated with FGF-2, heparin and IL-1b (Fig 6D) RT-PCR confirmed that S100A9 was not induced in primary murine fibroblast-like cells by FGF-2, heparin and IL-1b stimulants (not shown) Expression and regulation of S100A8 in fibroblasts Promoter analysis in 3T3 fibroblasts To examine mechanisms of transcriptional regulation of the S100A8 gene by IL-1b, and FGF-2 plus heparin, 5¢-flanking sequences upstream of the transcription initiation site, untranslated intron and sequences upstream of exon 1, were used to evaluate activities of deletion constructs after transient transfection into 3T3 cells (Fig 4) Marked differences between FGF-2heparin- and IL-1b-induced responses were seen with all constructs IL-1b did not change the luciferase activity of any construct although northern blotting of mRNA preparations of IL-1b-stimulated cells in the same experiment was positive (not shown) Levels of luciferase activity after FGF-2 ⁄ heparin stimulation were similar for all constructs, with two- to fourfold increases over luciferase activity in unactivated cells The region )94 to )34 bp contained the essential promoter because its deletion completely abrogated luciferase activity The region )178 to )94 bp is responsible for luciferase activity in unactivated cells (basal activity) because deletion strongly reduced basal activity but elements involved in FGF-2 enhancement were retained Consensus motifs for a number of transcription factors, including CCAAT ⁄ enhancer binding Fig Luciferase activity of S100A8 promoter deletion constructs in fibroblasts stimulated with FGF-2 + heparin or IL-1b Various S100A8 5¢-truncated mutant constructs shown on the left were used and the transcription initiation site was at position +1 Promoterless is the parent vector (pGL2) and Promoter is the promoterless with an SV40 promoter (pGL2-promoter) 3T3 cells were transiently transfected with various constructs and a Renilla luciferase construct The cotransfected cells were left unstimulated (in CM) or treated with FGF-2 (1.5 nM) + heparin (1 ImL)1) or IL-1b (10 mL)1) for 48 h The firefly luciferase activity, normalized to that of Renilla luciferase, was compared with the normalized activity of unstimulated promoter-transfected cells and expressed as normalized fold-induction The right bars represent averaged luciferase activities obtained from duplicates from at least three separate experiments Error bars represent standard deviation of the mean FEBS Journal 272 (2005) 2811–2827 ª 2005 FEBS 2815 Expression and regulation of S100A8 in fibroblasts F Rahimi et al protein (C ⁄ EBP), Ets, and E box are located within this region Constructs not containing the 1st exon and intron ()178–0 bp) generated positive, but relatively weak luciferase activities but in the same proportions, indicating that this region still contains elements essential for gene induction by FGF-2 A The MAPK pathway is involved in S100A8 mRNA induction by FGF-2 and IL-1b in 3T3 fibroblasts B The MAPK pathway is implicated in S100A8 mRNA induction by IFNc and LPS in macrophages [26] In 3T3 cells, PD 098059 (MAPK kinase (MEK) inhibitor, 50 lm) suppressed FGF-2- and FGF-2-heparininduced S100A8 mRNA by  65 and 62%, respectively, and SB 202190 [c-Jun N-terminal kinase (JNK) ⁄ p38 inhibitor, 10 lm] by  50% (Fig 5A) Similarly, PD 098059 (50 or 75 lm) or SB 202190 (10 or 20 lm) reduced responses to IL-1b by  70–83% (Fig 5B) indicating converging pathways in FGF-2- ⁄ heparin- and IL-1b-induced S100A8 mRNA induction in fibroblasts Cycloheximide completely abrogated the S100A8 gene in 3T3 cells stimulated with FGF-2 ± heparin (Fig 5C) or IL-1b (Fig 5D), indicating a requirement for de novo protein synthesis C D TGF-b suppresses FGF-2-induced mS100A8 mRNA in 3T3 and primary fibroblasts Because TGF-b modulates fibroblast function and phenotype [38,39], its effect on S100A8 gene expression was tested 3T3 cells cultured with TGF-b, heparin, or a combination of both, did not express S100A8 mRNA (Fig 6A) S100A8 mRNA induction by FGF-2 ± heparin was almost completely abrogated in cells simultaneously cultured with TGF-b for 24 h (Fig 6A) Quantitative RT-PCR showed 34.3-fold induction of S100A8 mRNA by FGF-2 ⁄ heparin which decreased approximately sixfold with TGF-b; mRNA levels induced by FGF-2 alone were halved in cells coincubated with TGF-b (Fig 6B) No S100A9 mRNA was found in any of the samples tested by RT-PCR in this experiment (not shown) In marked contrast to its effect on the FGF-2- ⁄ heparin-induced responses (lane 10, Fig 6C), TGF-b (8 pm-0.8 nm) did not reduce S100A8 mRNA levels induced by IL-1b (lanes 2–4, Fig 6C) However, the high mRNA levels with a combination of IL-1b, FGF2 and heparin (lane 5, Fig 6C) were reduced only by  74% with 0.08 nm TGF-b (lane 7, Fig 6C); concentrations up to 0.8 nm did not increase suppression (lane 8, Fig 6C) S100A8 mRNA induced with 2816 Fig Involvement of the MAPK pathways and de-novo protein synthesis in induction of S100A8 mRNA in fibroblasts (A) 3T3 cells stimulated (24 h) with FGF-2 (F, 1.5 nM) or FGF-2 and heparin (H, ImL)1) with ⁄ without 4-h preincubation with PD 098059 (PD, 50 lM) or SB 202190 (SB, 10 lM) as indicated (B) 3T3 cells stimulated (24 h) with IL-1b (10 mL)1) with ⁄ without 4-h preincubation with PD 098059 (50–75 lM) and SB 202190 (10–20 lM) Data represent three experiments (C) 3T3 fibroblasts stimulated with FGF-2 (F, 1.5 nM) and heparin (H, ImL)1) for 24 h with or without 4-h preincubation with lgỈmL)1 cycloheximide (CHX) as indicated C, mRNA from unstimulated cells; BM, murine bone-marrow RNA (D) 3T3 cells stimulated with IL-1b (10 mL)1) for 24 h with or without 4-h preincubation with cycloheximide (5 and 10 lgỈmL)1) as indicated Similar results observed in four experiments FGF-2 ⁄ heparin and IL-1b in BMF decreased by  67% with 0.08 nm TGF-b (Fig 6D) To clarify the mechanism of TGF-b suppression, 3T3 cells incubated for 20 h with FGF-2 ⁄ heparin in the presence ⁄ absence of TGF-b were treated with actinomycin D (ActD) for another 20 h to inhibit further transcription Cells stimulated with FGF-2 or FGF-2 ⁄ heparin harvested 20 h after treatment with FEBS Journal 272 (2005) 2811–2827 ª 2005 FEBS F Rahimi et al A Expression and regulation of S100A8 in fibroblasts A B B C Fig Expression of S100A8 protein in 3T3 cells (A) 20–25 · 106 confluent 3T3 cells unstimulated (S1, L1) or stimulated with 1.5 nM FGF-2 and ImL)1 heparin with (S2, L2) or without 10 mL)1 IL-1b (S3, L3) for  30 h Total cell-associated (L) and secreted (S) protein was immunoprecipitated, SDS ⁄ PAGE was in the presence of DTT (100 mM), and western analysis using anti-mOxA8 IgG Recombinant murine S100A8 (4, 40 and 100 ng) was the positive control Results obtained in two experiments (B) Approximately 20–25 · 106 cells were either unstimulated (lane 1) or stimulated (lane 2) for  30 h; cell lysates and supernatants (600 lL) were subjected to immunoaffinity purification through an anti-mS100A8 affinity column and C4 RP-HPLC The collected fractions were then analyzed by SDS ⁄ PAGE and western blotting in the absence of DTT Recombinant mS100A8 (25 ng) was used as the positive control D FGF-2 ⁄ heparin and treated with ActD were reduced by  49 and 66%, respectively, indicating reduced mRNA stability Similar results were obtained in two experiments (not shown) S100A8 protein in activated 3T3 fibroblasts Fig TGF-b regulates S100A8 mRNA induction by FGF-2 (A) Northern analysis of mRNA from 3T3 cells stimulated with FGF-2 (F, 1.5 nM), heparin (H, ImL)1), TGF-b (T, 0.08 nM) alone or in the combinations indicated for 24 h; C, unstimulated cells (B) RT-PCR analysis of the RNA samples used in (A) Levels of S100A8 mRNA were compared to those of HPRT in the corresponding samples and ratios of S100A8 mRNA ⁄ HPRT are indicated for each sample (C) Cells stimulated with FGF-2 (F, 1.5 nM) ⁄ heparin (H, ImL)1) with or without IL-1b (10 mL)1) in the presence or absence of increasing concentrations of TGF-b (8 pM-800 pM) as indicated BM, total bone-marrow RNA (D) Confluent BMF treated for 24 h with the indicated doses of FGF-2, heparin, IL-1b and TGF-b ActD had 126% of the mRNA levels of untreated cells, indicating somewhat increased mRNA stability ⁄ accumulation In the presence of TGF-b, mRNA levels from cells stimulated with FGF-2 or FEBS Journal 272 (2005) 2811–2827 ª 2005 FEBS S100A8 was not detected in unprocessed lysates or supernatants of 2.5–3 · 106 stimulated ⁄ unstimulated confluent 3T3 cells by western blot analyses or by double-sandwich ELISA (detection limit ¼ 0.03 nm [40]) Pull-down experiments, to concentrate the protein from a larger number of cells (20–25 · 106), showed no S100A8 in supernatants or lysates of unstimulated cells (S1 and L1, respectively, Fig 7A) Low levels were present in supernatants (S2, Fig 7A) but S100A8 was mainly cell-associated in cells stimulated for 30 h with FGF-2 ⁄ heparin and IL-1b (L2, Fig 7A) Separation was initially performed in the presence of dithiothreitol (DTT) to increase the yield by reducing disulfide-linked complexes and the characteristic monomeric mass of 10 kDa (rA8; Fig 7A) was confirmed In an alternative approach, supernatants and lysates of activated cells were concentrated using an affinity 2817 Expression and regulation of S100A8 in fibroblasts support and bound protein subjected to C4 reversephase HPLC Recombinant mS100A8 elutes as a single peak at 19.8–20 (not shown) Because no major peak were obtained (possibly due to low levels), fractions were collected between 17.25 and 21.25 (covering the expected retention-time range for native mS100A8 monomer and dimer), and lyophilized No mS100A8 was detected in fractions from supernatants (not shown) or lysates (lane 1, Fig 7B) of unstimulated cells Western blotting of the three fractions from lysates of stimulated cells collected over 18.25–20.25 (lane 2, fraction collected at 18.25–19.25 min, Fig 7B) contained components of molecular mass 20 kDa, with the same migration profile as dimeric mS100A8, contained in the positive control (lane 3, Fig 7B) No mS100A8 monomer (10 kDa) was detected This was unexpected as the same conditions have yielded monomeric and complexed forms of mS100A8 in other stimulated cell types [34,41] F Rahimi et al No S100A8 was found in unstimulated 3T3 cells stained with preimmune IgG or an antibody against HOCl-oxidized mS100A8 (mOxS100A8) (Fig 8A,B, respectively); 4,6-diamidino-2-phenylindole (DAPI)stained nuclei were evident Similarly, stimulated cells stained with the nonimmune IgG were unreactive (Fig 8C) Approximately 10–30% of 3T3 cells stimulated with FGF-2 ⁄ heparin ⁄ IL-1b showed bright cytoplasmic fluorescence (Fig 8D) When costimulated with TGF-b, S100A8-positive cells dropped to 5% of total (Fig 8E) Confocal microscopy clearly showed localization of S100A8 (red fluorescence) in the cytoplasm with no obvious associations with cytoskeletal structures (Fig 8F) S100A8 expression in rat dermal wounds To assess in vivo expression of S100A8 in fibroblasts, rat dermal wound tissue was used Examination of Fig Immunofluorescent detection of S100A8 Confluent 3T3 cells grown in slide chambers were unstimulated (A, B) or stimulated for 30 h with 1.5 nM FGF-2, ImL)1 heparin and 10 mL)1 IL-1b in the absence (C, D, F) or presence of 0.08 nM TGF-b (E) Permeabilized fixed cells stained with nonimmune rabbit IgG (A, C) or anti-mOxA8 (B, D, E, F) followed by antirabbit IgG-Alexa-Fluor-568 and the nuclear stain, DAPI, were analyzed by fluorescent ⁄ confocal microscopy A high-power image of a representative S100A8-positive fibroblast stimulated with FGF-2, heparin and IL-1b is shown (F) Similar results were obtained in two experiments Magnifications: (A–E) 400·; (F) 600· 2818 FEBS Journal 272 (2005) 2811–2827 ª 2005 FEBS F Rahimi et al wounded rat skin days post injury showed an intensely anti-S100A8-immunoreactive scab, containing S100A8-positive neutrophils (Fig 9Aa), impinging on the injured, and surrounding the normal epidermis In normal epidermis, the superficial more differentiated keratinocytes reacted with anti-mS100A8 more intensely than those in the stratum basale which contains the proliferating keratinocytes In the dermis, small dilated microvessels containing intensely stained Expression and regulation of S100A8 in fibroblasts S100A8-positive neutrophils were evident, representing the in situ positive controls (Fig 9Ab) Based on morphology, extravascular S100A8-positive cells were identified as macrophages or extravasating neutrophils Some spindle-shaped fibroblast-like cells closely apposed to, and aligned with collagen fibers were also relatively intensely mS100A8-positive (Fig 9Ab) although staining was heterogeneous These were identified as fibroblast-like cells, based on morphology, Fig Immunohistochemical localization of S100A8 in rat dermal wounds (A) Immunostaining of rat dermal wound days after injury (Aa) Low-power (200 ·) view of the wounded dermis beneath the neutrophil-rich S100A8-positive scab (S) Black arrows indicate S100A8-positive fibroblast-like cells apposed to collagen fibers indicated by C Neovessels and sebaceous glands are indicated by V and SG, respectively Delineated inset in (Aa) corresponds to (Ab) (400 ·) where neutrophils and macrophage-like cells are indicated by red and blue arrows, respectively Black arrows point to S100A8-positive fibroblast-like cells (B) Staining of rat wound days after injury Anti-S100A8 IgG (Ba, Bb) and nonimmune IgG (Bc) were used (Ba) The scab (S) is evident along the wounded epidermis and encroaching normal uninjured keratinocytes (K) Some hair follicles (H), small vessels (V) and sebaceous glands (SG) surrounded by collagen (C) fibers are evident (Bb) An area of granulation tissue showing macrophage-like (blue arrows) and fibroblast-like cells (black arrows) around neovessels (V) were S100A8positive Some S100A8-negative fibroblast-like cells are indicated by asterisks (Bc) Immunostaining of the 4-day wound with nonimmune IgG (C) Anti-mS100A8 immunostaining of rat wound days after injury (Ca) Low-power view of an area of mature granulation tissue and scar formation rich in collagen fibers and fibroblasts stained with anti-mS100A8 IgG The epidermal keratinocytes are evident to the right of the wounded area (K) The inset shown in (Ca) corresponds to (Cb) where S100A8-negative fibroblast-like cells are indicated by arrows FEBS Journal 272 (2005) 2811–2827 ª 2005 FEBS 2819 Expression and regulation of S100A8 in fibroblasts location in the granulation tissue and alignment with collagen fibers as no appropriate specific marker to detect rodent fibroblasts was available Similarly, in a mouse model of delayed-type hypersensitivity responses, about 10% of fibroblast-like cells were S100A8-positive (S Leong and C.L Geczy; unpublished data) In 4-day wounds, granulation tissue (macrophages, fibroblasts, neovessels and collagen) was well-established and most spindle-shaped fibroblast-like cells and macrophages stained positively with anti-mS100A8 (Fig 9Ba and b) Underneath the scab containing intensely stained S100A8-positive neutrophils, the uninjured and proliferating keratinocytes were S100A8positive; superficial cornified keratinocytes were more intensely positive, compared to controls (Fig 9Ba and c) In 7-day wounds, healing was evident, with little inflammatory component, decreased ⁄ no vascularity and many fibroblasts aligned along collagen fibers (Fig 9Ca) Fibroblast-like cells were S100A8negative and keratinocytes were weakly positive (Fig 9Cb) Discussion Fibroblasts are ubiquitous sentinel cells that participate in local inflammatory processes by directly interacting with infiltrating leukocytes Leukocytes residing in, or recruited into tissues are surrounded by a meshwork of fibroblasts and both cell types produce, and are influenced by mediators that regulate inflammation, wound healing, fibrosis, oxidative processes and vascular remodeling This study provides the first evidence that S100A8 is regulated in fibroblasts by particular growth factors, further supporting its role in inflammatory processes FGFs stimulate proliferation of many cell types involved in wound healing including endothelial cells, fibroblasts and keratinocytes and are essential for survival, replication, differentiation and migration of various cell types during embryonic and fetal development [42,43] Interestingly, S100A8 has roles in cell migration [24,25] and S100A8, but not S100A9 [29,44], is essential for embryonic development [45] FGF-2 up-regulated S100A8, but not S100A9, mRNA in 3T3 and primary fibroblasts A fragment of human S100A8 (S100A821)45) is chemotactic for fibroblast-like periodontal ligament cells [46] and this may be a function of S100A8 released as a consequence of wound healing and in an inflammatory environment S100A8 induction was confluence-dependent (Fig 1), a requirement similar to its induction in microvascular endothelial cells [34] Although fibroblasts not normally 2820 F Rahimi et al establish contacts in vivo, in culture they establish gap junctions [37] indicating metabolic interdependence Cell–cell ⁄ cell–ECM contacts may provide additional signals for optimal induction of the S100A8 gene in vitro and because of the potentiation exhibited by heparin, ECM heparan sulfate may be important FGFs bind heparin and heparan sulfates in positively charged pockets that facilitate formation and stabilization of FGF-2 dimers and higher oligomers, resulting in a more stable FGF–FGFR signaling complex [47] Heparin also protects FGF against glycosylation [48] and proteolysis and FGF binding to ECM increases its immediate availability and durability of action [47] Responses to FGF-2 were strongly up-regulated and prolonged by heparin (Figs 1–3 and 6), but heparin alone had no effect (Fig 6A), implying facilitation at the level of the FGF–FGFR interaction IL-1b has high amino-acid homology with FGF-2, and is a potent inducer of the S100A8 gene in microvascular endothelial cells [34] and weak inducer in macrophages [26] IL-1b up-regulated S100A8 mRNA in fibroblasts (Fig 3) and exceptionally strong synergy was observed when in combination with FGF-2 ⁄ heparin, obvious at the mRNA (Table 1) and protein level (Figs and 8) In contrast, LPS, IFNc and TNF, which induce S100A8 in macrophages [26] and endothelial cells [34] only induced S100A8 mRNA when used in combination, suggesting cooperative pathways IL-1b synergistically increased S100A8 mRNA in primary fibroblasts costimulated with FGF-2 ⁄ heparin (Figs 3D and 6D) Because of functional and phenotypic heterogeneity [36,37], fibroblasts from various sources were tested and because responses in primary cells were relatively weak, quantitative RT-PCR was used to detect the S100A8 gene and to assess S100A9 gene coinduction The stability of S100A8 protein in neutrophils is suggested to be dependent on S100A9 coexpression [29], but, like the situation in activated murine macrophages [26,27] and keratinocytes [22], S100A9 was not coinduced with S100A8 in any fibroblast type tested, strongly supporting our proposal that S100A8 ⁄ S100A9 coexpression is not mandatory for the function or stability of murine S100A8 This may not be the case with the human proteins that are generally coexpressed [49] although S100A8 alone was detected in the human LDL proteome by mass spectrometry and peptide mass fingerprinting [28] Negligible S100A8 was secreted in response to IL-1b and FGF-2 ⁄ heparin (Fig 7A) This is in stark contrast with activated macrophages which secrete high levels in response to various stimulants, particularly LPS with IL-10 or prostaglandin E2 [26,27], but similar to IL-1-activated microvascular endothelial cells [34] and FEBS Journal 272 (2005) 2811–2827 ª 2005 FEBS F Rahimi et al UVA-irradiated keratinocytes [22], where the protein is located in the nuclei and cytoplasm of a proportion of cultured cells The high levels of S100A8 mRNA found in FGF-2 ⁄ heparin-activated fibroblasts costimulated with IL-1b correlated well with protein levels detected by western blotting (Fig 7), and S100A8-positive fibroblasts were more numerous (Fig 8) S100A8 induced by FGF-2 ⁄ heparin ⁄ IL-1b was mainly cytoplasmic (Fig 8F) with no obvious differences in distribution between cells with apparently pyknotic or normal nuclei FGF-2 signals through the MAPK and extracellular signal-regulated kinase (ERK1, p44) and ERK2 (p42) pathways [43] Similarly, IL-1b activates MAPK and stress-activated protein kinases which are involved in the G0–G1 cell-cycle transition in human neuroma fibroblasts [50] IL-1b can also activate p38 MAPK, protein kinases A and C (PKC), phospholipase C, and signals via nuclear factor jB [51] The MAPK pathway is important in cell proliferation, differentiation, embryogenesis and synthesis of inflammatory cytokines [43,52] and is essential for S100A8 induction in macrophages [27] Inhibitors of the MEK and p38 pathways both partially abrogated FGF-2 ⁄ heparin- and IL-1b-induced expression in 3T3 fibroblasts (Fig 5), indicating their partial involvement Induction of S100A8 mRNA by FGF-2 was dependent on de novo protein synthesis (Fig 5) FGF-2 signaling can also occur via activation of phospholipase C-c, PKC and increased Ca2+ flux [43] The S100A8 gene is also regulated by PKC- and Ca2+-dependent mechanisms in macrophages [26,53], and these results suggest complex control involving converging pathways for its induction in fibroblasts Promoter deletion analyses indicated differential regulation of S100A8 induction by IL-1b and FGF2 ⁄ heparin (Fig 4) The minimal promoter required for FGF-2- ⁄ heparin-induced responses was restricted to the region from )94 to +465 (Fig 4) Deletion of the region from )178 to )94 bp, which contains activated protein-1, Ets and C ⁄ EBP motifs, totally negated activity induced by LPS ⁄ IL-10 in macrophages [27], indicating distinct responsive elements activated by FGF-2 ⁄ heparin in fibroblasts and LPS ⁄ IL-10 in macrophages IL-1b-responsive elements may be distinct from FGF-2-responsive elements as they were not located within )917 to +465 bp and preliminary experiments with a construct spanning 4.9 kb upstream of the transcriptional start site strongly indicate differences in transcriptional regulation The strong synergy generated by the combination of FGF-2 ⁄ heparin and IL-1b may be mediated through distinct enhancer ⁄ responsive elements in the two regions or IL-1b may enhance S100A8 mRNA stability FEBS Journal 272 (2005) 2811–2827 ª 2005 FEBS Expression and regulation of S100A8 in fibroblasts TGF-b accelerates deposition, remodeling and maturation of collagen later in the healing process of dermal wounds [54,55] Differentiation of fibroblasts from a proliferative phenotype to collagen-producing and then to myofibroblastic phenotypes is believed to be mediated by TGF-b [39] IL-1b and FGF-2 promote proliferation [56,57], whereas TGF-b promotes differentiation to myofibroblasts [39] and antagonizes the IL-1- and FGF-2-induced proliferative phenotypes [58,59] Here we show that S100A8 mRNA (Fig 6) and protein (Fig 7) induced by FGF-2 ⁄ heparin was suppressed in fibroblasts costimulated with TGF-b, possibly by decreasing S100A8 mRNA stability Interestingly, induction by IL-1b was unaltered (Fig 6) The effect is similar to TGF-b inhibition of the FGF2-induced aA-crystallin promoter activity observed in lenticular epithelial explants and TGF-b has been associated with pathological changes, including apoptosis and accumulation of ECM, in some forms of cataract [60] The pattern of S100A8 gene regulation indicates that this protein may be involved in fibroblast-to-myofibroblast differentiation at sites of inflammation and repair ⁄ remodeling, particularly as S100A8 has been associated with myeloid cell differentiation [26,61] Moreover, S100A8 and S100A9 are expressed in human and murine wounds and are associated with keratinocyte proliferation ⁄ differentiation [21] Whether the TGF-b-induced myofibroblast phenotype correlates with down-regulation of S100A8 is worthy of investigation, particularly as S100A8 expression in monocytes disappears in tissue [61] and exudate macrophages [26] These investigations are underway in our laboratory During the later stages of wound healing, TGF-b is active and its receptors highly expressed whereas FGF-2 activity decreases [54,55] At this stage, fibroblast-derived S100A8 would be expected to be downregulated This would be consistent with reduced inflammation in the growing scar tissue and less oxidative stress as levels of tissue antioxidants including glutathione, catalase, superoxide dismutase, glutathioneS-transferase, and glutathione peroxidase partially or completely recover as healing progresses [62] This correlated well with the immunohistochemical studies in the healing rat wound Some spindle-shaped fibroblast-like cells in the granulation tissue were S100A8positive and days after dermal injury whereas almost all fibroblasts were negative days post injury (Fig 9) The increasing importance of various S100 proteins in regulating oxidative processes is emerging [33] and in the mouse, S100A8, but not S100A9, was induced in dermal keratinocytes following oxidative stress and by ultraviolet A irradiation, and gene 2821 Expression and regulation of S100A8 in fibroblasts induction was dependent on generation of ROS [22] Interestingly, under nonreducing ⁄ nondenaturing conditions, only mS100A8 dimer was detected in activated fibroblast lysates (Fig 7B), a structural modification induced in S100A8 by oxidants such as peroxide [31], suggesting a function partially analogous to S100A2 [63] S100A2 also forms disulfide bonds in response to H2O2 and is sensitive to early cellular responses to oxidative stress [33,63] Redox-dependent signaling and intracellular levels of ROS regulate cell-densitydependent growth [64,65] and increasing fibroblast confluence concurs with augmented intracellular oxidative stress [65] Interestingly, in lung fibroblasts, FGF-2 rapidly increases intracellular O2– without extracellular H2O2 release whereas TGF-b promotes extracellular H2O2 release that is O2– independent and NADHoxidase- ⁄ Ras-independent [66] Because S100A8 is an efficient scavenger of ROS [30,31] it may regulate intracellular redox-mediated pathways involved in fibroblast growth ⁄ differentiation These pathways are currently under investigation in our laboratory Experimental procedures Reagents Recombinant mS100A8 was generated from the glutathione-S-transferase fusion protein expressed in Escherichia coli as described previously [67] For production of polyclonal antibodies, S100A8 and mOxS100A8 (50 lg) [30] bound to nitrocellulose particles in Freund’s complete adjuvant (Sigma, St Louis, MO, USA) were prepared as described [67], and injected intradermally into New Zealand white rabbits Rabbits were boosted after and weeks with 100 lg mOxS100A8 or mS100A8 in incomplete Freund’s adjuvant (Sigma) IgG from pooled sera was purified by Protein A-Sepharose (Amersham Pharmacia Biotech, Buckinghamshire, UK) Titer and reactivities of IgG with mS100A8 ⁄ mOxS100A8 were tested by immunoblotting and ELISA as described [40] Pre-immune sera did not react with mS100A8 or mOxS100A8 Anti-mOxS100A8 and antimS100A8 did not cross-react with S100A9 monomer but recognized the murine and rat S100A8 monomer and oxidized forms from 20 kDa to 90–97% To determine whether de novo protein synthesis was required for induction of S100A8 mRNA, confluent 3T3 cells were preincubated with the protein synthesis inhibitor, cycloheximide (5–10 lgỈmL)1), for h before stimulation with activators and RNA analysis performed after 24 h For inhibition of RNA synthesis, fibroblasts were stimulated with particular stimulants for 20 h before an additional 20-h incubation with ActD (10 lgỈmL)1) and RNA levels analyzed before and after ActD addition To determine the role of the MAPK pathway in S100A8 mRNA induction, 3T3 cells were preincubated for h with inhibitors (PD 098059, the MEK inhibitor, 50–75 lm; SB 202190, JNK ⁄ p38 inhibitor, 10–20 lm) before stimulation RNA extraction and Northern analysis Total cellular RNA from 2.5 to 3.5 · 106 fibroblasts was purified as described [69] Murine BM RNA [67] was used as a positive control RNA (25–30 lg) was sizefractionated on 1.5% agarose gels containing 0.4 lgỈmL)1 ethidium bromide and transferred onto Hybond N+ membranes (Amersham) [69] Pre-hybridizations were performed at 53 °C (2 h) in a formamide-containing buffer [67] 32P-labelled mS100A8 riboprobe and 18S rRNA oligoprobe were prepared [34] and purified using ProbeQuant G-50 Micro Columns (Amersham) Blots were hybridized at 53 °C overnight, washed [34] and exposed to phosphoimager plates (Bio-Rad or FUJI Photo Film Co, Ltd, Japan) Blots were scanned using the Molecular Imager System (GS-525, Bio-Rad) and analyzed with Multianalyst 1.0 software (Bio-Rad) Alternatively, the FLA5000 imaging system (FUJI) and the L Process and Image Gauge software packages were used The relative magnitude of expression of S100A8 mRNA was FEBS Journal 272 (2005) 2811–2827 ª 2005 FEBS Expression and regulation of S100A8 in fibroblasts determined using quantitative densitometry, normalized to the level of 18S rRNA on the same blot and expressed as the percent maximum mRNA level In some experiments, total RNA was subjected to quantitative RT-PCR using the primers described [6] cDNA levels during the linear amplification phase were normalized against hypoxanthine phosphoribosyl-transferase (HPRT) controls Assays were in triplicate and mean ± standard deviation (SD) determined Reporter assays The mS100A8-promoter-luciferase-fused reporter plasmids were described previously [27] Sub-confluent 3T3 cells grown in 24-well plates were transiently transfected with 0.8 lg firefly luciferase constructs, or the parent plasmid pGL2-basic DNA, or the pGL2 promoter DNA combined with 0.03 lg of the control pRL-TK plasmid (Promega) in the presence of LipofectAMINE After 24 h, cells were replenished with fresh DMEM CM and stimulated with FGF-2 (1.5 nm) + heparin (1 ImL)1) or with IL-1b (10 mL)1) for another 48 h Firefly and Renilla luciferase activities in cell extracts (20 lL) were assayed using the Dual-Luciferase Assay System in a TD20 ⁄ 20 Luminometer (Turner Design, Sunnyvale, CA, USA) following manufacturer’s instructions Promoter activity was normalized to Renilla luciferase which resulted in reproducible and constant values relative to pGL2 promoter Protein purification and Western blotting Control or stimulated (28–30 h) 3T3 cells (20–25 · 106) were washed twice with DPBS after removal of supernatants, lysed in DPBS containing 1% (v ⁄ v) Triton X-100, 50 mm Tris ⁄ HCl, pH 8.0 and Complete Protease Inhibitors, subjected to three freeze-thaw cycles and sonicated on ice for Debris was removed by centrifugation and lysates (5 mL) and supernatants (9 mL) concentrated to ⁄ 5th starting volume in a vacuum lyophilizer Following dialysis overnight at °C in water (1 L) to remove excess salt, samples were incubated with anti-mOxS100A8 (10 lgỈmL)1) overnight at °C, then with agarose-bound caprine anti-rabbit IgG slurry (70 lLỈmL)1) overnight at °C Beads were sedimented (1600 g), washed thrice with lysis buffer, boiled (100 °C, min) in Tricine sample buffer (50 lL, Bio-Rad), and 20 lL separated by SDS ⁄ PAGE as described previously [67], with DTT (100 mm) Alternatively, supernatants and lysates were directly adsorbed with Sepharose-coupled anti-mS100A8, washed with phosphatebuffered saline (NaCl ⁄ Pi) and bound proteins eluted with 0.2 m glycine ⁄ m NaCl (pH 3.0; 0.5 mL), and pH adjusted with 100 lL Tris ⁄ HCl (pH 9.0) Eluates (600 lL) were directly subjected to C4 reverse-phase HPLC [31] prior to nonreducing SDS ⁄ PAGE and Western blotting [67] 2823 Expression and regulation of S100A8 in fibroblasts Protein localization Confluent 3T3 cells in Lab-Tek Permanox chamber slides (Nalge Nunc International, Roskilde, Denmark) were activated with stimulants for 30 h, rinsed thrice in NaCl ⁄ Pi, slides air-dried then re-hydrated in NaCl ⁄ Pi (10 min), fixed in 4% paraformaldehyde (10 min) and permeabilized (10 min) with 0.5% saponin ⁄ 0.1% BSA in NaCl ⁄ Pi After blocking with normal goat serum (20% w ⁄ v in saponin– BSA solution, 20 min), and incubation with antimOxS100A8 IgG or preimmune rabbit IgG (both at 10 lgỈmL)1 in saponin–BSA) overnight at °C, slides were rinsed in NaCl ⁄ Pi and incubated with anti-rabbit IgGAlexa-Fluor-568 (1 : 200, in saponin–BSA) for h in the dark at 25 °C, rinsed in NaCl ⁄ Pi (3 · min) and nuclei stained with DAPI (0.3 nm in NaCl ⁄ Pi, 10 in the dark) Slides were mounted in Vectashield Mounting Medium (Vector Laboratories, Burlingame, CA, USA), cover-slipped and examined using a Leica DM IRB inverted confocal microscope attached to a Leica TCS SP scanner (Leica Microsystems, Mannheim, Germany) Images were captured using Leica Confocal Software and processed using Adobe Photoshop 7.0 For immunohistochemistry, paraffin-embedded tissue blocks of dermal wounds from three rats wounded by scalpel incision under anesthesia, sutured and dressed, were provided by Professor Rolfe Howlett Rats were sacrificed 2, and days after wounding and fixed tissue processed, sectioned (5 lm) and mounted onto silane-coated slides; all experiments were conducted according to local ethics committee approval Deparaffinized sections were re-hydrated then washed in NaCl ⁄ Pi ⁄ Triton X-100 (0.3% v ⁄ v, 15 min), treated with 0.2 m HCl (15 min), permeabilized with proteinase K (25 lgỈmL)1) in 0.1 m Tris ⁄ HCl ⁄ 50 mm EDTA, pH 8.0 (37 °C, 30 min), washed with 0.2% (w ⁄ v) glycine followed by NaCl ⁄ Pi, and fixed with 4% paraformaldehyde in NaCl ⁄ Pi (5 min) Sections were blocked sequentially with H2O2 (3% v ⁄ v in NaCl ⁄ Pi, 30 min) and 10% normal goat serum (30 min), 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3919–3928 2827 ... Titer and reactivities of IgG with mS100A8 ⁄ mOxS100A8 were tested by immunoblotting and ELISA as described [40] Pre-immune sera did not react with mS100A8 or mOxS100A8 Anti-mOxS100A8 and antimS100A8... stimulated with FGF-2, heparin and IL-1b (Fig 6D) RT-PCR confirmed that S100A9 was not induced in primary murine fibroblast- like cells by FGF-2, heparin and IL-1b stimulants (not shown) Expression and regulation... Expression and regulation of S100A8 in fibroblasts Promoter analysis in 3T3 fibroblasts To examine mechanisms of transcriptional regulation of the S100A8 gene by IL-1b, and FGF-2 plus heparin, 5¢-flanking