Pentoxifylline, Pentifylline, and Interferons Decrease Type I

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Pentoxifylline, Pentifylline, and Interferons Decrease Type I and III Procollagen mRNA Levels In• Dermal Fibroblasts: Evidence for Mediation by Nuclear Factor 1 Down-Regulation

Matthew R. Duncan, Anthony Hasan, and Brian Berman Department of Dermatology and Cutaneous Surgery, University of Miami School of Medicine, Miami, Florida, U.S.A.

Pentoxifylline (PTX) is a methylxanthine that exhib steady-state levels of the corresponding pro collagen its multiple biologic activities, including the inhibi mRNA transcripts. Reduction of pro collagen mRNA tion of collagen synthesis by dermal fibroblasts. Be levels appeared to be dependent on new protein cause some PTX activities have recently been linked synthesis, as it was prevented by treatment with to transcription factor-mediated regulation of gene cycloheximide. Assay for the presence of nuclear transcription, we have investigated if PTX acts to NF-l by gel mobility shift analysis showed that ex inhibit collagen synthesis at a transcriptional locus by tracts from interferon, PTX, and PTF-treated fibro measuring pro collagen mRNA levels and by assaying blasts lacked proteins recognizing the consensus for the presence of an activator of pro collagen gene DNA binding sequence for NF-1. Taken together, promoters, nuclear factor (NF)-1. The effects of an these observations suggest interferons and methylx other methylxanthine, pentifylline (PTF) , shown anthines may inhibit fibroblast collagen synthesis by herein to be a tenfold more potent inhibitor of col a common mechanism requiring new protein synthe lagen synthesis than PTX, and interferon-a, -(3, and sis that suppresses pro collagen gene transcription -11 were studied in parallel. Analysis of extracellular through down-regulation of NF-1. Key words: cyto protein and RNA from 48-h-treated fibroblasts kines/extracellular matrix/transcription factors. J Invest Der showed that PTX, PTF, and interferons decreased matol 104:282-286, 1995 al(I), a2(I), and al(III) pro collagens by reducing the

entoxifylline (PTX) is a substituted methylxanthine genome contain binding sequences for the transcnptlOn factor long used for the treatment of intermittent claudication NF-KB, whose activation is inhibited by PTX treatment [9,10]. and other conditions involving defective regional mi We have previously reported that PTX may also have therapeu crocirculation. This hemorheologic effect of PTX is tic potential as an antifibrotic agent, as PTX inhibits the collagen thought to be mediated by inhibition of cyclic nucle and glycosaminoglycan production of normal dermal, sclerodermal, otide phosphodiesterases resulting in increased erythrocyte flexibil and keloidal fibroblasts [11,12]. Our original observations have ity and the production by platelets and vascular endothelial cells of recently been confIrmed by others and extended to fibroblasts prostaglandins that enhance local blood flow and promote throm derived from other cOlmective-tissue disorders involving excess bolysis [1]. More recently PTX has been shown to have potential as extracellular matrix deposition [13,14]. To determine if PTX a treatment for several cytokine-mediated diseases, including irri inhibits collagen syntheses by transcription-related mechanisms we tant and contact hypersensitivity, AIDS, transplant-related toxicity, treated normal dermal fibroblasts with PTX and assayed the levels and septic shock via inhibition of tumor necrosis factor (TNF)- a of mRNA transcripts for type I and III procollagen a-chains by production or action [2-7]. Regulation of gene transcription ap Northern blotting and assessed the presence of a pro collagen pears to be the primary locus for PTX inhibition of TNF-a gene-activating transcription factor, NF-1, by a gel mobility shift production and action as PTX decreases levels of mRNA for assay [15,16]. The effects of PTX treatment were compared to TNF-a in endotoxin-treated monocytes and macrophages, mRNA those caused by the collagen-synthesis-inhibitory cytokines, inter for TNF-a-induced proteins, such as collagenase and 2' -5' oligo feron (IFN)-a, -{3, -'Y, and a more potent substituted methylxan adenylate synthetase in fibroblasts, and human immunodeficiency thine collagen-synthesis inhibitor, pentifylline (PTF) [17,18]. Our virus long-terminal repeat-directed gene expression in infected results indicate that PTX, PTF, and IFN s all decrease pro collagen cells [6,8,9]. Both the enhancer region of the TNF-a gene and the synthesis, pro collagen mRNA levels, and the presence of NF-l. long-terminal repeat region of the human immunodeficiency virus MATERIALS AND METHODS Reagents PTX and PTF were obtained from Sigma Chemical Co., St. Manuscript received April 8, 1994; revised August 30, 1994; accepted for Louis, MO. Recombinant human IFN-0'2b' -{3, and -T' derived from publication October 11, 1994. Escherichia coli were obtained from Schering, Kenilworth, NJ, Janssen Abbreviations: NF-l, nuclear factor-I; PTF, pentifylline; PTX, pentoxi Biochirnica, Flanders, NJ; and Genentech, South San Francisco, CA, fylline. respectively. Plasmids Hf667, Hf32, and Hf934 containing eDNA inserts

282 VOL. 104, NO.2 FEBRUARY 1995 PENTOXlFYLLlNE INHmITS PROCOLLAGEN mRNAs 283 complementary to mRNAs for human pro collagen a1 (I), a2(I), and a1 (III), RESULTS respectively, were from the American Type Culture Collection, Rockville, MD, and a 1.0-kb cDNA probe for human glyceraldehyde-3-phosphate PTX and Other Xanthine Analogues Decrease Fibroblast dehydrogenase (GAPDH) was supplied by Clontech Inc., Palo Alto, CA. Collagen Production Although our previous in vitro studies Goat anti-type I collagen IgG was from Fisher BioTech, Pittsburgh, PA, and indicate PTX may be a potential antifibrotic agent, the high murine monoclonal IgG1 to human type III collagen from Chemicon concentrations of PTX necessary to suppress fibroblast collagen International, Temecula, CA. PTX, PTF, and IFN s diluted to desired synthesis suggest that routine oral doses of PTX would be ineffec concentrations with Dulbecco's modified Eagle's medium (DMEM) con tive as an antifibrotic therapy [11 ,12]. To address this problem, we taining 0.1 % human serum albumin were added directly to fibroblast compared the collagen synthesis-inhibitory potency of PTX and cultures. several other subtituted methylxanthines using the fibroblast mi Fibroblast Cultures Primary fibroblast cultures were initiated by the crowell collagen assay in an attempt to fmd a more potent explant technique from full-thickness adult facial and mammary skin collagen-synthesis inhibitory xanthine. As shown in Fig 1, caffeine removed during cosmetic surgery and maintained in DMEM containing 25 (1 ,3 ,7-trimethylxanthine) , theophylline (1 ,3-dimethylxanthine) , mM HEPES, 2 mM glutamine, 100 Vlml penicillin, 100 J.Lg/ml streptomy theobromine (3,7 -dimethylxanthine) and 3-isobutyl-l-methylxan cin, and 10% heat-inactivated fetal bovine serum (FBS) (Whittaker Bio thine all inhibited fibroblast collagen synthesis with a 50% inhibi products, Walkersville, MD) at 37°C in a 5% CO humidified atmosphere. 2 tory dose (IDso) of approximately 100 p,g/ml, similar to that of Fibroblasts were subcultured by trypsinization and studies were performed PTX (1-[5-oxohexyl]-3,7-dimethylxanthine). The parent com on cells between the third and ninth passages. pound, xanthine, was insoluble at 1000 p,g/ml but showed no Assay of Fibroblast Collagen Production Collagen produced during inhibition of collagen synthesis at 100 p,g/ml, whereas hypoxan the terminal 24 h of a 48-h treatment period by steady-state, confluent thine had an IDso greater than 1000 p,g/ml. In contrast, the PTX fibroblast microcultures in DMEM plus ascorbic acid and no FBS was precursor compound, PTF (1-hexyl-3, 7 -dimethylxanthine), was assessed by 3H-proline incorporation into pepsin-resistant, salt-precipitated found to be ten times more potent than PTX inhibiting fibroblast extracellular and cell-associated collagen as we and others have previously collagen synthesis with an IDso of approximately 10 p,g/ml. described [8,11,12,18]. Each experimental condition was done in triplicate wells and averaged results are expressed as disintegrations per minute (dpm) Western Blot Analyzed Extracellular Collagen Production 3 of 3H-collagen per 10 cells ~ SD. Harvested fibroblasts were always Is Decreased by IFNs9 PTX9 and PTF To confirm the validity greater than 95% viable by trypan blue exclusion. of our results obtained by the 3H-proline incorporation assay, and to determine the collagen types inhibited by PTX and PTF we Gel Electrophoresis and Western Blotting Aliquots of protease analyzed fibroblast supernatant media for types I and III collagens inhibitor treated supernatant media from unradiolabeled microcultures were by immunoblot analysis. The effects of IFN-a, -{3 and -yon types electrophoresed on 12% sodium dodecylsulfate-polyacrylamide gels after I and III collagen synthesis was also assessed for comparison. We reduction with 2-mercaptoethanol using modified Laemmli conditions [19]. Supernatants were concentrated 20 times for analysis of collagens and found that the only proteins recognized by antibodies to types I and aliquot volumes were standardized for cell number. Following semi-dry III collagens migrated with molecular weights of approximately transfer to neutral nylor. membrane, Western immunoblots were prepared 160-170 kD, indicating that fibroblast supernatant media contained by incubation with primary antibodies to collagen type I or III and predominantly full-length procollagens. The polyclonal antibody to secondary alkaline phosphatase-conjugated antibodies, followed by detec type I collagen reacted preferentially with the lower-molecular tion with a chemiluminescent dioxetane substrate system (Rad-Free, Schlei weight a2 chain of procollagen I. As shown, in Fig 2, treatment cher & Schuell, Keene, NH). Blot signals detected on x-ray film were with 100 ng/ml ofIFNs, 1000 p,g/ml PTX, or 100 p,g/ml PTF for quantified by densitometry. 48 h decreased the amount of both types I and III procollagens present in supernatant media from normal dermal fibroblasts. All RNA Isolation and Northern Blotting Total cellular RNA was ex tracted from fibroblast monolayers grown in 25-cm2 flasks by a single-step agents decreased both the al and a2 chains of type I procollagen. method of RNA isolation [20] with a commercially available acid guani The percentage decrease induced by IFN-a, -{3, -y, PTX, and PTF dinium thiocyanate-phenol extraction reagent (Tri Reagent, Molecular as assessed by densitometry are for procollagen I, 34, 49, 59, 84, Research Center, Cincinnati, OH). Five-microgram aliquots of RNA were and 76, respectively; and for procollagen 111,51,16,65,97, and 97, glyoxylated and fractionated on a 1.0% agarose gel in 10 mM phosphate respectively. Given that fibroblasts produce pro (I) and (III) buffer, pH 7, followed by vacuum blotting onto a nylon membrane (Nytran, Schleicher & Schuell) using methods similar to those previously described [21]. Membranes were hybridized overnight at 42°C against cDNA probes labeled by random priming with digoxigenin-modified dVTP [22] in a hybridization solution containing 50% formamide, 5 X SSPE, 1 X Den ~ 150 • hardt's solution, 100 J.Lg/ml denatured salmon sperm DNA, and 0.1 % .... sodium dodecylsulfate. Following low- and high-stringency washes signals gc;;- were generated by incubation with an anti-digoxigenin antibody coupled to 0(1) o °100 alkaline phosphatase and a chemiluminescent dioxetane substrate (Genius 0:'" System, Boehringer-Mannheim, Indianapolis, IN) [22]. Hybridization sig a..~ nals detected on x-ray film were quantitated by densitometry and are z reported relative to the expression of the "housekeeping gene," GAPDH WE ~ .g. 50 [23]. The proal(I) collagen probe was random primed from the 1.8-kb -oJ- EcoRI/EcoRI fragment of plasmid Hf677 [24], the proa2(I) collagen probe -oJ from the 2.2-kb AatII/EcoRI fragment of plasmid Hf32 [25], and the o U proal(III) collagen probe from the 0.7- and 0.6-kb EcoRI/HindIII frag ments of plasmid Hf934 [26]. OL---~------~------~--______~~ 10 100 1000 Gel Mobility Shift Assay The binding of nuclear proteins extracted XANTHINES (Jlglml) from 25-cm2 cultures of confluent fibroblasts to a digoxigenin-labeled 22-mer double-stranded oligonucleotide containing the consensus binding Figure 1. PTX and other xanthine analogues decrease normal sequence for NF-l (5'-TGGCANsGCCAA-3') (Promega, Madison, WI) dermal fibroblast collagen production. Collagen production was as was determined by resolution of oligonucleotide-protein complexes from sessed by measuring 3H-proline incorporation into cellular and extracellular free oligonucleotide by native polyacrylamide gel electrophoresis as we pepsin-resistant, salt-precipitated collagen during the terminal 24 h of a 48-h have previously described in detail, with the following modifications [27]: treatment with xanthines as described in Materials and Methods. PTF (t-. ), the binding assay mixture contained 3 J.Lg of nuclear protein extract and poly PTX (.), caffeine (0), theophylline (_), theobromine (0), isobutylmeth dG:dC in place of poly dI:dC in a total volume ofl0 J.LI; and electrophoresis ylxanthine (e), hypoxanthine ( 0 ), xanthine ( . ). Points are the mean of and pre-electrophoresis were performed at 200 V with recirculation of the triplicate determinations and SD were less than 15%. Data was confirmed in buffer. two additional experiments. 284 DUNCAN ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOG)

1 234 5 6 1 2 3 456

Procollagen I Procollagen al(l)

Procollagen III GAPDH Figure 2. IFNs, PTX, and PTF decrease extracellular type I and III procollagen. Supernatant media from fibroblast microcultures were har Figure 4. IFNs, PTX, and PTF do not decrease levels of pro colla- I vested after 48 h treatment and analyzed by Western immunoblot analysis. gen al(l) mRNA in fibroblasts additionally treated with 2 ILg/ml of Untreated fibroblasts (1), fibroblasts treated with 100 nglml ofIFN-a (2), cycloheximide. Treatments as described in Fig 2. 100 ng/ml IFN-t3 (3), 100 nglml IFN-'}' (4), 1000 J-Lg/ml PTX (5), or 100 J-Lg/ml PTF (6).

IFNs, PTX, and PTF Decrease Fibroblast Expression of collagens in a ratio of 5:1, then the observed decreases are NF-1 NF-l is a nuclear protein that has been implicated as a approximately proportional to the decrease in total collagen syn transcription factor required for basal and transforming growth thesis determined by the 3H-proline incorporation assay shown in factor (TGF-f3)-stimulated procollagen gene promoter activation ! Fig 1, and reported previously [11-13,17,18]. Moreover, PTX and [15,16,28,29]. To determine if IFN, PTX, and PTF inhibition of PTF at the tested concentrations appear to be more effective pro collagen mRNA levels can be correlated with reduced NF-l inhibitors of collagen synthesis than IFNs at 100 ng/ml. expression, we analyzed nuclear protein extracts from untreated fibroblasts and fibroblasts treated for 48 h with IFNs, PTX, or PTF IFNs, PTX, and PTF Decrease Fibroblast Procollagen for the presence of factors binding an oligonucleotide containing mRNA Levels To determine if IFN, PTX, and PTF-inhibited the consensus binding sequence for NF-l using the gel mobility procollagen synthesis is due to decreased steady-state pro collagen shift assay [27]. As shown in Fig 5, nuclear extracts from untreated mRNA levels, Northern blot analysis was performed using RNA fibroblasts (lane 2) contain proteins that bind a portion of the extracted from dermal fibroblasts that were either untreated or labeled NF-l consensus oligonucleotide, resulting in the detection treated with IFNs, PTX, or PTF for 48 h. Blots were incubated of a DNA-protein complex of shifted mobility that is composed of with cDNA probes that hybridized to mRNA transcripts of 5.8 and labeled oligonucleotide bound to protein NF-l. In contrast to 4.8 kb for proal (I) collagen, 5.1 and 4.6 kb for proa2(1) collagen, untreated fibroblasts, nuclear extracts from IFN-a, -f3, -y, PTX, or 5.4 and 4.8 kb for proal(llI) collagen, and 1.4 kb for the PTF-treated fibroblasts (lanes 3-7) produced no complexes of "housekeeping protein" GAPDH [23-26]. The resultant Northern shifted mobility indicating the absence of proteins in the extracts of blots shown in Fig 3 demonstrate that the steady-state levels of all treated fibroblasts that interact with NF-l binding sites. As shown three assayed procollagen mRNA transcripts were decreased by in Fig 6, the shifted DNA-protein complexes appear to be specific treatment with IFN s, PTX, or PTF. Only minimal effects on for NF-l protein as simultaneous incubation with a lO X concen GAPDH transcripts were noted, with IFN s causing small decreases tration of unlabeled NF-l oligonucleotide prevents detection of and PTX and PTF small increases in transcript levels. When the shifted labeled oligonucleotide-protein complexes using nuclear levels of pro collagen mRNA transcripts are corrected for these extracts from untreated, IFN-a-treated, and PTX-treated fibro slight changes in GAPDH transcript expression, the percentage blasts. Furthermore, the shifted DNA-protein complex appears to decrease in procollagen mRNA levels induced by IFNa, -f3 , -y, be specific for NF-l protein, as we have previously demonstrated PTX, and PTF as assessed by densitometry are, for pro collagen that simultaneous incubation with a lOX concentration of unla 0'1(1), 60, 60, 80, 95, and 95, respectively; procollagen 0'2(1), 14, beled NF-l oligonucleotide prevents detection of shifted oligonu 33,52,64, and 80, respectively; and pro collagen 0'1(111), 31, 33, 71, cleotide-protein complex [27], whereas unrelated oligonucleotides 92, and 98, respectively. When IFN-a, -f3, -y, PTX, and PTF were SP-l and AP-2 do not prevent detection (data not shown). Direct tested in the presence of 2 J.Lg/ml of protein synthesis inhibitor addition of IFN s, PTX, or PTF to binding reactions between cycloheximide, no decrease in procollagen 0'1 (I) mRNA transcripts was observed (Fig 4). 1234567 123456

Procollagen al (I) Oligo-protein Procollagen a2(1) Complex

Procollagen al (III) Free Oligo

GAPDH Figure 5. IFN, PTX, and PTF-treated fibroblasts lack proteins binding a NF-l consensas oligonucleotide. Nuclear extracts from Figure 3. IFNs, PTX, and PTF decrease levels of type I and III untreated fibroblasts (2) or fibroblasts treated for 48 h with IFN-a (3), -13 procollagen a-chain mRNA transcripts. Fibroblasts treatment as de (4), -'}' (5), PTX (6), or PTF (7) at the concentrations listed in Fig 2. scribed in Fig 2. Procollagen transcripts were detected with cDNA probes Fibroblast nuclear protein extracts were incubated with labeled NF-l prepared from plasmids Hf677 [al(I)], Hf32 [a2(I)], and Hf943 [al(III)]. consensus oligonucleotide, followed by electrophoresis, blotting, and de Detections were performed simultaneously with a cDNA probe for GAPDH tection as described in Materials and Methods. Lane 1 is labeled NF-l mRNA transcripts as described under Northern Blotting. consensus oligonucleotide without nuclear extract. VOL. 104, NO.2 FEBRUARY 1995 PENTOXIFYLUNE INHIBITS PROCOLLAGEN rnRNAs 285

1 34 5 binding, extracts from fibroblasts treated with IFN-a, -{3, - )I, PTX, 2 6 and PTF lacked oligonucleotide-binding proteins (Fig 5), suggest ing these agents inhibit fibroblast collagen synthesis by reducing the presence of nuclear NF-1. Whether nuclear extracts contain no NF-1 due to enhanced degradation, inhibited. synthesis, or transfer to the cytoplasm or whether NF-l is present but in an inactive form that does not bind the oligonucleotide cannot be determined from Oligo-protein our gel mobility shift analysis. Modulation of NF-1 synthesis or degradation may be the more likely hypothesis, as to date NF-1 has Complex not been reported to exist in an inactive form nor to undergo nucleus-cytoplasm translocation [33]. However, both PTX and IFNs have been shown to mediate the expression of other genes through the regulation of transcription factor activation [9,10,34]. Overall our results suggest that IFNs and methylxanthines may Free Oligo act through a common mechanism to inhibit fibroblast collagen synthesis, i.e., inhibition of procollagen mRNA levels by suppres Figure 6. Excess u nlabeled NF-l oligonucleotide specifically sion of procollagen gene transcription through down-regulation of blocks IFN- an d P TX-treated fibroblast pro teins from binding to transcription factor NF-1. This theory is supported by experiments labeled NF-l co nsensus oligonucleotide. Nuclear extracts from un showing that when IFNs and PTX are tested in combination, the treated fibroblasts (1 and 4) or fibroblasts treated for 48 h with IFN-a (2 and inhibitory effects on collagen synthesis are only additive and not 5) or PTX (3 and 6) at the concentrations listed in Fig 2. Fibroblast nuclear potentiating (data not shown). Moreover, our results showed that protein extracts were incubated with labeled NF-1 consensus oligonucleo tide in the presence (1, 2, and 3) and absence (4, 5, and 6) of a tenfold PTF, PTX, and other tested methylxanthines inhibit fibroblast concentration of unlabeled oligonucletide, followed by electrophoresis, collagen synthesis in relative potency to their activities as nonspe blotting, and detection as described in Materials and Methods. cific cyclic nucleotide phosphodiesterase inhibitors [35]. Inhibition of phosphodiesterases can affect gene transcription, as increased cyclic adenosine monophosphate and cyclic GMP levels activate protein kinase A and G, which activate latent proteins, including labeled oligonucleotide and nuclear extracts from untreated fibro transcription factors, by serinelthreonine phosporylation [36]. blasts did not prevent oligonucleotide-protein complex formation, Likewise, early reports also indicate that IFN-a/ {3 can also increase indicating these agents do not physically block NF-1-DNA inter the levels of cyclic AMP and cyclic GMP in cultured cells, and more action (data not shown). recently IFN-a, -(3 and -)I have been reported to activate gene promoter regulatory proteins through tyrosine kinase-mediated DISCUSSION phosphorylation [34,37]. Thus, the common mechanism by which IFNs-a, -{3, -)I and PTX are agents that we and others have IFNs and PTX/PTF inhibit fibroblast collagen synthesis likely previously reported to decrease the collagen synthesis of fibroblasts involves the interaction of mixed kinase signaling pathways result derived from both normal and fibrotic skin [11-13,17,18]. In this ing in phosphorylation of a common protein mediator, which report we have demonstrated that other methylxanthines also ultimately leads to down-regulation of NF-1. inhibit fibroblast collagen production and that one compound, PTF, is a tenfold more potent inhibitor than PTX. Although several reports indicate that IFN-)I inhibits fibroblast collagen production r¥e thank Bonnie Posey for helpful secretarial assistana in preparing this manuscript, by decreasing both basal and TGF-{3-stimulated levels of procolla Dr. Robin Bergerfor assistance with th e gel mobility shift assay, and Genentech, Inc. gen mRNAs through both transcriptional and post-transcriptional for supplying IFN-y. process, the mechanisms by which IFN-a, -{3, and PTX inhibit This J/lork was supported ill part by a Merit R eview Grant from the Department collagen synthesis have not been widely studied [29-32]. 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