FEBS 18753
`
`FEBS Letters 410 (1997) 259-264
`
`Post-transcriptional regulation of inducible nitric oxide synthase mRNA
`in murine macrophages by doxycycline and chemically modified
`tetracyclines
`Ashok R. Amina,b,c,d,*, Rajesh N. Patela, Geeta D. Thakkera, Charles J. Lowenstein°,
`Mukundan G. Attura, Steven B. Abramsona,b
`"Department of Rheumatology and Medicine, Hospital for Joint Diseases, 301 East 17th Street, New York, NY 10003, USA
`bDepartment of Medicine, New York University Medical Center, 550 First Avenue, New York, NY 10016, USA
`'Department of Pathology, New York University Medical Center, 550 First Avenue, New York, NY 10016, USA
`dKaplan Cancer Research Center, New York University Medical Center, 550 First Avenue, New York, NY 10016, USA
`'Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
`
`Received 8 May 1997
`
`Abstract Chemically modified tetracyclines [CMT-3 (IC50
`-6-13 JIM= -2.5-5 J.lglml) and CMT-8 (ICso -26 J.1M =
`10 Jlglml), but not CMT-1, -2 or -51, which lack anti-microbial
`activity, inhibited nitrite production in LPS-stimulated macro-
`phages. Unlike competitive inhibitors of L-arginine which
`inhibited the specific activity of inducible nitric oxide synthase
`(iN OS) in cell-free extracts, CMTs exerted no such direct effect
`on the enzyme. CMTs could, however, be shown to inhibit both
`iNOS mRNA accumulation and protein expression in LPS-
`stimulated cells. Tetracyclines (doxycycline and CMT-3) unlike
`hydrocortisone had no significant effect on murine macrophages
`transfected with iN OS promoter (tagged to a luciferase reporter
`gene) in the presence of LPS. However, doxycycline and CMT-3
`augmented iNOS mRNA degradation, in LPS-stimulated murine
`macrophages. These studies show a novel mechanism of action of
`tetracyclines which harbours properties to increase iN OS mRNA
`degradation and decrease iNOS protein expression and nitric
`oxide production in macrophages. This property of tetracyclines
`may have beneficial effects in the treatment of various diseases
`where excess nitric oxide has been implicated in the pathophys-
`iology of these diseases.
`© 1997 Federation of European Biochemical Societies.
`
`Key words: Nitric oxide; Macrophages; Tetracycline;
`Chemically modified tetracyclines (CMT); Collagenase; RNA
`
`1. Introduction
`
`Nitric oxide (NO), first identified as an endothelium-derived
`relaxation factor, is now recognized to regulate the functions
`of many mammalian cells and tissues [!]. NO is produced by
`the ubiquitous enzyme, nitric oxide synthase (NOS). The over-
`expression of inducible NOS (iNOS) in a variety of inflamma-
`tory tissues had led many to conclude that the modulation of
`NO synthesis and action could represent a new approach to
`treatment of inflammatory and autoimmune diseases [2,3],
`including osteoarthritis (OA) [4] and rheumatoid arthritis
`(RA) [5].
`
`*Corresponding author. Fax: (1) (212) 598 6168.
`
`Abbreviations: CMT, chemically modified tetracycline; GAPDH,
`glyceraldehyde-3-phosphate dehydrogenase; LPS, lipopolysaccharide;
`MMP, matrix metalloprotease; NO, nitric oxide; NOS, nitric oxide
`synthase; iNOS, inducible nitric oxide synthase; OA, osteoarthritis;
`RA, rheumatoid arthritis
`
`0014-5793/97/$17.00 © 1997 Federation of European Biochemical Societies. All rights reserved.
`Pl1 soo 14-579 3 (97)00605 -4
`
`Various studies have shown that among the tetracycline
`group of broad-spectrum antibiotics, doxycycline and mino-
`cycline exert biological effects independent of their anti-micro-
`bial activity [6-9]. Such effects include inhibition of activity of
`matrix metalloproteases
`(MMPs),
`including collagenase
`(MMP-1), gelatinase (MMP-2) and stromelysin (MMP-3),
`and prevention of pathogenic tissue destruction [6]. Recent
`studies have also indicated that tetracyclines and inhibitors
`of MMPs block tumor progression [10], bone resorption [11]
`and angiogenesis [12]. In view of these diverse effects of tetra-
`cyclines, we have also observed that doxycycline and minocy-
`cline inhibit iNOS expression in murine macrophages [13].
`The present study shows that: (a) some chemically modified
`tetracyclines (CMT-3 and CMT-8, but not CMT-1 or CMT-5)
`share the ability to inhibit iNOS expression in a similar fash-
`ion as observed with doxycycline and minocycline; (b) CMT-3
`and doxycycline augment iNOS mRNA degradation and have
`no significant influence on iNOS gene transcription.
`
`2. Materials and methods
`
`Murine macrophage cells (RAW 264.7) were obtained from ATCC
`(Rockville, MD, USA). An anti-murine iNOS antibody was obtained
`from Transduction Laboratories (Lexington, KY, USA). Doxycycline,
`minocycline, hydrocortisone, N-acetyl imidazole (NAI), and lipopoly-
`saccharide (LPS) were ohtained from Sigma (St. Louis, MO, USA).
`The CMTs (designated as CMT-1, -2, -3, -5 and -8) were a generous
`gift from CollaGenex (Newtown, PA, USA).
`Equal amounts of protein (25-50 ~g) estimated by BCA reagent
`(Pierce, Rockford, IL, USA) were loaded onto SDS-PAGE gels and
`stained to verify the concentrations of various protein fractions by
`examining the intensities of the protein bands on the gels. Western
`blot analysis was carried out from the same cell extracts. The Western
`blot was probed with a specific anti-iNOS murine rnAb as specified by
`Transduction Laboratories. Membranes with bound antibodies (e.g.,
`iNOS) were stripped by submersion in stripping buffer (100 mM
`2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.7) and incu-
`bating at 50°C for 30 min with occasional agitation. Membranes were
`then washed twice for 10 min at RT using large volumes of wash
`buffer. The same blot was also probed with an anti-actin antibody
`generously provided by Dr. James L. Lessard (Children's Hospital
`Medical Center, Cincinnati, OH, USA) using the standard protocol.
`Blots were developed using the ECL Western blot system (Amersharn,
`Arlington Heights, IL, USA). Quantitation of the bands was per-
`formed using a densitometer from Molecular Dynamics (Sunnyville,
`CA, USA).
`Total RNA was isolated using TRI Reagent (MRC, Cincinnati,
`OH, USA). Northern blot analysis was carried out as described earlier
`[14,15]. Briefly, 20 flg of RNA was subjected to electrophoresis in 1%
`agarose formaldehyde gel, and th
`Dr. Reddy's Laboratories, Ltd., et al.
`v.
`Galderma Laboratories, Inc.
`IPR2015-__
`Exhibit 1025
`
`

`
`FEBS 18753
`
`FEBS Letters 410 (1997) 259-264
`
`Post-transcriptional regulation of inducible nitric oxide synthase mRNA
`in murine macrophages by doxycycline and chemically modified
`tetracyclines
`Ashok R. Amina'bc'd'*, Rajesh N. Patela, Geeta D. Thakkera, Charles J. Lowenstein0,
`Mukundan G. Attura, Steven B. Abramsona'b
`aDepartment of Rheumatology and Medicine, Hospital for Joint Diseases, 301 East 17th Street, New York, NY 10003, USA
`bDepartment of Medicine, New York University Medical Center, 550 First Avenue, New York, NY 10016, USA
`cDepartment of Pathology, New York University Medical Center, 550 First Avenue, New York, NY 10016, USA
`dKaplan Cancer Research Center, New York University Medical Center, 550 First Avenue, New York, NY 10016, USA
`"Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
`
`Received 8 May 1997
`
`(IC50
`|CMT-3
`tetracyclines
`Abstract Chemically modified
`~ 6 - 13 uM = ~ 2.5-5 jig/ml) and CMT-8 (IC50 ~ 26 uM =
`10 (ig/nil), but not CMT-1, -2 or -5], which lack anti-microbial
`activity, inhibited nitrite production in LPS-stimulated macro-
`phages. Unlike competitive
`inhibitors of L-arginine which
`inhibited the specific activity of inducible nitric oxide synthase
`(iNOS) in cell-free extracts, CMTs exerted no such direct effect
`on the enzyme. CMTs could, however, be shown to inhibit both
`iNOS mRNA accumulation and protein expression in LPS-
`stimulated cells. Tetracyclines (doxycycline and CMT-3) unlike
`hydrocortisone had no significant effect on murine macrophages
`transfected with iNOS promoter (tagged to a luciferase reporter
`gene) in the presence of LPS. However, doxycycline and CMT-3
`augmented iNOS mRNA degradation, in LPS-stimulated murine
`macrophages. These studies show a novel mechanism of action of
`tetracyclines which harbours properties to increase iNOS mRNA
`degradation and decrease iNOS protein expression and nitric
`oxide production in macrophages. This property of tetracyclines
`may have beneficial effects in the treatment of various diseases
`where excess nitric oxide has been implicated in the pathophys-
`iology of these diseases.
`© 1997 Federation of European Biochemical Societies.
`
`Key words: Nitric oxide; Macrophages; Tetracycline;
`Chemically modified tetracyclines ( C M T ); Collagenase; R NA
`
`1. Introduction
`
`Nitric oxide (NO), first identified as an endothelium-derived
`relaxation factor, is now recognized to regulate the functions
`of many mammalian cells and tissues [1]. NO is produced by
`the ubiquitous enzyme, nitric oxide synthase (NOS). The over-
`expression of inducible N OS (iNOS) in a variety of inflamma-
`tory tissues had led many to conclude that the modulation of
`NO synthesis and action could represent a new approach to
`treatment of inflammatory and autoimmune diseases [2,3],
`including osteoarthritis
`(OA) [4] and rheumatoid arthritis
`(RA) [5].
`
`*Corresponding author. Fax: (1) (212) 598 6168.
`
`tetracycline; GAPDH,
`Abbreviations: CMT, chemically modified
`glyceraldehyde-3-phosphate dehydrogenase; LPS, lipopolysaccharide;
`MMP, matrix metalloprotease; NO, nitric oxide; NOS, nitric oxide
`synthase; iNOS, inducible nitric oxide synthase; OA, osteoarthritis;
`RA, rheumatoid arthritis
`
`tetracycline
`Various studies have shown that among the
`group of broad-spectrum antibiotics, doxycycline and mino-
`cycline exert biological effects independent of their anti-micro-
`bial activity [6-9]. Such effects include inhibition of activity of
`matrix metalloproteases
`(MMPs),
`including
`collagenase
`(MMP-1), gelatinase (MMP-2) and stromelysin
`(MMP-3),
`and prevention of pathogenic tissue destruction [6]. Recent
`studies have also indicated that tetracyclines and
`inhibitors
`of M M Ps block tumor progression [10], bone resorption [11]
`and angiogenesis [12]. In view of these diverse effects of tetra-
`cyclines, we have also observed that doxycycline and minocy-
`cline inhibit iNOS expression in murine macrophages [13].
`The present study shows that: (a) some chemically modified
`tetracyclines (CMT-3 and CMT-8, but not CMT-1 or CMT-5)
`share the ability to inhibit iNOS expression in a similar fash-
`ion as observed with doxycycline and minocycline; (b) CMT-3
`and doxycycline augment iNOS m R NA degradation and have
`no significant influence on iNOS gene transcription.
`
`2. Materials and methods
`
`Murine macrophage cells (RAW 264.7) were obtained from ATCC
`(Rockville, MD, USA). An anti-murine iNOS antibody was obtained
`from Transduction Laboratories (Lexington, KY, USA). Doxycycline,
`minocycline, hydrocortisone, ./V-acetyl imidazole (NA1), and lipopoly-
`saccharide (LPS) were obtained from Sigma (St. Louis, MO, USA).
`The CMTs (designated as CMT-1, -2, -3, -5 and -8) were a generous
`gift from CollaGenex (Newtown, PA, USA).
`Equal amounts of protein (25-50 u.g) estimated by BCA reagent
`(Pierce, Rockford, IL, USA) were loaded onto SDS-PAGE gels and
`stained to verify the concentrations of various protein fractions by
`examining the intensities of the protein bands on the gels. Western
`blot analysis was carried out from the same cell extracts. The Western
`blot was probed with a specific anti-iNOS murine mAb as specified by
`Transduction Laboratories. Membranes with bound antibodies (e.g.,
`iNOS) were stripped by submersion in stripping buffer (100 mM
`2-mercaptoefhanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.7) and incu-
`bating at 50°C for 30 min with occasional agitation. Membranes were
`then washed twice for 10 min at RT using large volumes of wash
`buffer. The same blot was also probed with an anti-actin antibody
`generously provided by Dr. James L. Lessard (Children's Hospital
`Medical Center, Cincinnati, OH, USA) using the standard protocol.
`Blots were developed using the ECL Western blot system (Amersham,
`Arlington Heights, IL, USA). Quantitation of the bands was per-
`formed using a densitometer from Molecular Dynamics (Sunnyville,
`CA, USA).
`Total RNA was isolated using TRI Reagent (MRC, Cincinnati,
`OH, USA). Northern blot analysis was carried out as described earlier
`[14,15]. Briefly, 20 ug of RNA was subjected to electrophoresis in 1%
`agarose formaldehyde gel, and then transferred via capillary action
`
`0014-5793/97/S17.00 © 1997 Federation of European Biochemical Societies. All rights reserved.
`P / / S 0 0 1 4 - 5 7 9 3 ( 9 7 ) 0 0 6 0 5 -4
`
`Exh. 1025
`
`

`
`260
`
`A.R. Amin et al.lFEBS Letters 410 (1997) 259-264
`
`onto a nylon membrane (Zeta Probe, Bio-Rad Laboratories, Melville,
`NY, USA). The membrane was hybridized with [32P]dCTP-labelled
`iNOS cDNA (4 kb Smal fragment), a kind gift from Dr. James Cun-
`ningham (Harvard Medical School, Boston, MA, USA). After hybrid-
`ization, the blot was exposed to Kodak X-ray film (Kodak, Rochester,
`NY, USA) for 24-48 h with intensifying screens at -70°C. The
`GAPDH probe was purchased from ClonTech (Palo Alto, CA,
`USA) and probed as described above. Quantitation of the intensity
`of the iNOS and GAPDH bands was performed using a Personal
`Densitometer SI (Molecular Dynamics).
`Specific activity of iNOS was determined in cell-free extracts by
`monitoring the conversion of L-[3H]arginine to L-[3H]citrulline as de-
`scribed [15,16], RAW 264.7 cells were induced with LPS (100 ng/ml)
`in the presence and absence of minocycline, CMTs or hydrocortisone
`for 14-20 h. Following induction, the cells were pelleted at 4°C and
`resuspended in Tris buffer (10 mM, pH 7.4) containing 10 pg/ml each
`chymostatin, antipain, leupeptin and pepstatin, 1 mM DTT and 1 mM
`PMSF [15]. Cells were lysed in a Polytron PT1200 homogenizer (Kin-
`ematica, Switzerland) after 3 cycles of rapid freeze-thawing. The lysate
`was centrifuged at 16000 rpm for 60 min at 4°C, and the supernatants
`were used as cell-free extracts. The protein was measured by BCA
`assay reagent using BSA as standard [17]. The reaction mixture for
`iNOS assay consists of Tris 50 mM (pH 7.8); BSA 1 mg/ml; DTT
`1 mM; CaCl2 2 mM; FAD 10 pM; BH4 10 pM; L-arginine 30 uM;
`NADPH 1 mM. The reaction mixture was spiked with 1 pi (250 nM)
`of L-[3H]arginine (Du Pont NEN, Boston, USA, MA) (1 mCi/
`ml = 37.0 MBq/ml). After 20 min the assays were terminated by heat-
`ing the reaction mixture at 90°C for 5 min; 10 pi ( = 100000 cpm) of
`the supernatant was spotted on activated Avicel TLC plates (Anal-
`tech, Newark, DE, USA). The TLC plates were developed in a solvent
`system consisting of ethanol/water/ammonia (80:16:4). Quantitation
`of the spot for L-[3H]citrulline was performed by a Bioscan System
`200 Imaging Scanner.
`RNA stability analysis was carried out as previously described [18].
`Briefly, cells were stimulated with 100 ng/ml of LPS (± tetracycline)
`followed by addition of actinomycin D (5 pg/ml) 4 h post-stimulation.
`Total RNA was prepared at different time periods and analyzed by
`Northern blot analysis as described above. The data are represented
`as percentage of iNOS mRNA degraded after normalizing the values
`with GAPDH.
`RAW 264.7 cells were transfected with the 1.7 kb murine iNOS
`promoter linked to a luciferase reporter gene [19] using the DEAE-
`dextran method as previously described [20]; 10 pg of the plasmid
`DNA was added to 107 cells in 1.0 ml of DMEM (without serum)
`containing DEAE-dextran (250 pg/ml) and 50 mM Tris (pH 7.4). The
`suspension was further incubated at 37°C for 60 min followed by a 1.0
`min shock with 10% DMSO at room temperature. These cells were
`incubated for 45 min with the respective drugs and stimulated with
`100 ng/ml LPS 24 h post-transfection. The cells were finally harvested
`24 h after LPS stimulation and assayed for luciferase activity as de-
`scribed by the manufacturer's instructions (Promega, Madison, WI,
`USA).
`
`Doxycycline
`
`Minocycline
`
`CH
`3 OH
`H
`
`H
`
`N(CH3)2
`
`lT%T
`
`HO
`
`O
`
`1 0
`^CNH.
`
`^%y
`y
`1 II
`OH
`O
`
`OH
`
`(CH3)2N
`
`N(CH3)2
`
`H
`
`H
`
`rf^Y
`
`- ^y
`Y
`. OH I
`OH
`O
`
`HO
`
`O
`
`1 0
`CNH.
`
`H,C„
`
`-OH
`
`CMT-1
`
`CMT-2
`
`CMT-3
`
`CMT-5
`
`CMT-8
`
`HO
`
`O
`
`OH
`
`O
`
`NMe,
`
`HO
`
`O
`
`OH
`
`O
`
`OH
`
`O
`
`HO
`
`N
`
`NH
`
`OH
`
`HX
`
`3. Results and discussion
`
`CNH,
`
`Our recent studies show that doxycycline and minocycline
`inhibit iNOS expression at the level of iNOS mRNA accumu-
`lation in murine macrophages stimulated with LPS [13]. In
`view of these observations, we examined if chemically modi-
`fied tetracyclines as shown in Fig. 1 and which lack anti-mi-
`crobial activity as previously reported [21], could also modu-
`late iNOS expression. We therefore compared the effects of
`CMTs and minocycline on nitrite accumulation in LPS-stimu-
`lated murine macrophages as shown in Fig. 2. CMT-3 >
`CMT-8 (but not CMT-1, -2, or -5) inhibited nitrite accumu-
`lation in a dose-dependent manner. The IC50 level of CMT-3
`was < 2.5 pg/ml (< 6 uM) while that of CMT-8 was ~ 10 pg/
`ml ( ~ 26 uM). CMT-2 and CMT-5 at concentrations of 10
`pg/ml did not show a significant effect on nitrite accumulation
`(Fig. 2), whereas a marginal effect (inhibition) was seen with
`minocycline and CMT-1 at similar concentrations. These ex-
`
`Fig. 1. Structures of doxycycline, minocycline and various chemi-
`cally modified tetracyclines used in this study (provided by Colla-
`genex, Inc.).
`
`periments demonstrate that CMT-3 is more effective in its
`ability to inhibit nitrite accumulation than doxycycline, min-
`ocycline [13], and other CMTs tested in these studies.
`We further examined the effects of CMT-3 and CMT-8 on
`the enzyme activity of iNOS. Cells were stimulated with LPS
`in the presence of equal amounts of CMT-3 or -8 for 16 h.
`The medium was assayed for nitrite and iNOS enzyme in cell
`extracts in an L-arginine-to-L-citrulline conversion assay. As
`expected, CMT-3 and hydrocortisone inhibited nitrite accu-
`mulation significantly more than CMT-8. These data were
`substantiated by a significant decrease in specific enzyme ac-
`
`Exh. 1025
`
`

`
`A.R. Amin et al.lFEBS Letters 410 (1997) 259-264
`
`261
`
`□
`■
`^
`
`0 (ng/ml)
`2.5 (ng/ml)
`5.0 (ng/ml)
`
`[ Hi 0.0 (jlg/ml)
`
`Uninduced P1
`
`CMT 1
`
`C M T2
`
`iiiiiiiiiiiiiiiiniiiiiiiiiiiiiiriiiiirii-
`
`mm ^^m
`
`C M TS
`
`TTTTTTMI] nnn ni; nnn ii Inn ilii m i—
`
`0
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`liM Nitrite
`
`Fig. 2. Effect of CMTs on nitrite accumulation in murine macrophages stimulated with LPS. Murine macrophages (RAW 264.7 cells) were
`stimulated with LPS (100 ng/ml) in the presence of 10 |xg/ml minocycline (MINO) and various concentrations of CMTs in triplicate for 48 h.
`The levels of nitrite were estimated by the modified Griess method [31]. Data are expressed as |a,M nitrite accumulated of triplicate determinants
`(n = 3). Statistics were derived using unpaired Student's /-test. Data represent 1 of 3 similar experiments.
`
`tivity of iNOS in the intact cells, as shown in Table 1. These
`experiments demonstrated that the decrease in nitrite accumu-
`lation by the CMTs could be partially to the decrease in iNOS
`enzyme activity within the cells.
`Various investigators have shown that tetracyclines inhibit
`collagenase activity via direct effects on the enzyme [22-24].
`Another mechanism proposed for this phenomenon is that
`procollagenase is reduced to inactive fragments upon activa-
`tion in the presence of doxycycline [23]. We have recently
`shown that acetylating agents such as aspirin and N-acetyl
`imidazole [15], as well as competitive inhibitors of L-arginine
`(NMA), inhibit iNOS catalytic activity in cell-free extracts. In
`view of these observations, we examined the direct effect of
`CMTs on the ability of iNOS to convert radiolabelled L-
`[3H]arginine
`to L-citrulline
`in cell-free extracts
`in vitro.
`RAW 264.7 cells were stimulated with LPS for 16 h and
`cell-free extracts were made. Separate aliquots of equal
`amounts of enzyme were preincubated with various concen-
`trations of CMTs (6-24 uM), NMA (200 uM) or NAI (1 mM)
`for 20 min before the enzyme reaction was initiated after the
`addition of co-factors. As expected, NMA and NAI showed
`
`75 and 45% inhibition of iNOS enzyme activity, respectively,
`but there was no significant effect (< 5% inhibition) by the
`CMTs (data not shown). These experiments demonstrate that
`the action of these CMTs, like the doxycycline and minocy-
`cline [13], seems to be distinct from those reported for MMPs
`such as procollagenase at similar concentrations [22,23,25]. In
`view of the above observation and our previous experiments
`which indicate that iNOS protein is decreased by doxycycline
`and minocycline [13], we tested the effects of CMT-3 and —8
`at various concentrations on iNOS protein expression. RAW
`264.7 cells were stimulated with LPS in the presence and ab-
`sence of CMTs for 16 h; cell-free extracts were prepared and
`examined for iNOS expression by Western blot analysis. Fig.
`3 shows that, like minocycline [13], CMT-3 > CMT-8 inhib-
`ited 133 kDa iNOS expression. The effect of CMT-3 at 2.5 u.g/
`ml was similar to that observed with 20 u.g/ml of minocycline.
`The inhibition in the accumulation of nitrite in the same ex-
`periment was substantiated with the data shown in the West-
`ern blot analysis. Hydrocortisone, as expected, inhibited iNOS
`expression as previously reported [15]. Furthermore, there was
`no significant difference in the constitutively expressed pro-
`
`Table
`Effect of CMTs on the specific activity of iNOS
`Modulating agent
`Nitrite released
`Specific activity
`% inhibition
`% inhibition
`pmol/min per mg protein
`uM
`N/A
`Control (uninduced)
`<0.1
`<5
`N/A
`N/A
`LPS induced
`22.9
`90.8
`N/A
`CMT-3 (10 |ig/ml)
`47
`12.2
`37.6
`58.6
`CMT-8 (10 ng/ml)
`26
`17.0
`79.2
`12.7
`Hydrocortisone (10 uM)
`11.8
`51.5
`43.3
`49
`Murine macrophage cells were stimulated with 100 ng/ml of LPS in the presence of CMTs or hydrocortisone for 16 h. The nitrite accumulated in
`the medium was examined and the enzyme activity assayed from cell-free extracts. Percent inhibition of nitrite/specific activity was calculated after
`comparing the values with LPS-stimulated cells. 10 ug/ml of CMT-3/8 was equivalent to 27 uM. The data represent one of two similar experiments.
`N/A, not applicable.
`
`Exh. 1025
`
`

`
`262
`
`A.R. Amin et allFEBS Letters 410 (1997) 259-264
`
`Fig. 3. Western blot analysis of iNOS in RAW 264.7 cells exposed to minocycline, CMTs and hydrocortisone in the presence of LPS for 16 h.
`Mino: represents minocycline (20 ug/ml equivalent to 40 u,M). Hydro: represents hydrocortisone (10 uM equivalent to 3.4 ug/ml). CMT-3 (2.5,
`5 and 10 |tg/ml equivalent to 27, 13 and 6 |xM, respectively) and CMT-8 (5 and 10 (xg/ml equivalent to 27 and 13 |xM, respectively). Cell-free
`extracts were prepared and aliquots were blotted and probed with a specific a-iNOS mAb. The percent inhibition of iNOS expression was com-
`pared to LPS-stimulated cells, as determined by a densitometer scan, after normalizing the values with P-actin in the same blot. The upper pan-
`el shows the representative nitrite values in this particular experiment. The data represent 1 of 2 similar experiments.
`
`teins (such as P-actin) when the same blot was reprobed with
`anti-P-actin antibodies (Fig. 3). These experiments indicate
`that the decrease in NOS activity could be due to a decrease
`in the expression of iNOS protein.
`Based on our previous studies with doxycycline and mino-
`cycline, we tested the ability of CMTs to inhibit iNOS mRNA
`accumulation in RAW 264.7 cells stimulated with LPS for 16
`
`h. Fig. 4 shows a dose-dependent inhibition of iNOS mRNA
`accumulation by CMT-3. CMT-8 at 5 and 10 ug/ml showed a
`significant decrease in iNOS mRNA accumulation as com-
`pared to LPS-stimulated cells, whereas CMT-1 at 10 (j.g/ml
`showed no significant effect when the values were normalized
`with the respective GAPDH signals. The effect of hydrocorti-
`sone in this particular experiment was relatively less when the
`
`Fig. 4. Northern blot analysis of iNOS mRNA expression in RAW 264.7 cells stimulated with LPS in the presence of CMTs at 16 h. Total
`RNA was extracted and analyzed by Northern blot using a-iNOS and a-GAPDH probes. The iNOS/GAPDH signal was quantitated using a
`phosphoimager. The percent inhibition of iNOS mRNA expression was normalized with the GAPDH signal and compared with the values of
`the LPS-stimulated cells. Data represent 1 of 2 similar experiments.
`
`Exh. 1025
`
`

`
`A.R. Amin et allFEBS Letters 410 (1997) 259-264
`
`263
`
`LPS
`
`LPS+Doxycycline
`
`LPS+CMT-3
`
`150
`120
`90
`60
`30
`0
`Minutes after addition of Actinomycin D
`Fig. 5. Effect of doxycycline and CMT-3 on iNOS mRNA stability.
`RAW 264.7 cells were stimulated with 100 ng/ml of LPS in the
`presence or absence of doxycycline (40 ng/ml) or CMT-3 (10 |ig/ml)
`for 4 h, followed by addition of actinomycin D (5.0 |xg/ml). The
`cells were then harvested at different time intervals (0-120 min) and
`analyzed for iNOS mRNA accumulation as described in Section 2.
`The values for iNOS mRNA accumulation were normalized with
`GAPDH for equal loading. The percent inhibition of iNOS mRNA
`in tetracycline-treated cells was calculated against LPS-stimulated
`cells alone using a densitometer.
`
`mRNA was in its steady state [15]. The effects of hydrocorti-
`sone have been seen to be more significant when the iNOS
`mRNA is examined at 4 h post-stimulation where it blocks
`N F - KB activation and iNOS transcription.
`To examine the mechanism by which tetracyclines (doxycy-
`cline and CMT-3) decrease iNOS RNA accumulation, we
`evaluated the effects of these compounds on iNOS transcrip-
`tion and mRNA stability. Murine macrophages were trans-
`fected with a full-length iNOS promoter (tagged to a lucifer-
`ase reporter gene) and stimulated with LPS in the presence
`and absence of doxycycline, CMT-3 and hydrocortisone.
`Stimulation of these cells with LPS alone increased the rela-
`tive light units of luciferase activity from 0.22 to 35.0 which
`was designated as 100%. In a parallel experiment, 10 uM of
`hydrocortisone showed 88% inhibition of luciferase activity.
`Doxycycline at 20 and 40 ug/ml (41 and 82 uM, respectively)
`and CMT-3 at 10 ug/ml (25 uM) which markedly inhibited
`nitrite accumulation had no significant effect on the luciferase
`activity in the presence of LPS (data not shown). These ex-
`periments indicate that, unlike hydrocortisone, tetracyclines
`have no significant effect on iNOS transcription when stimu-
`lated with LPS.
`We next examined the effects of tetracyclines on iNOS
`mRNA stability. Murine macrophage cells were stimulated
`with LPS (± doxycycline or CMT-3) for 4 h followed by in-
`cubation with actinomycin D. Total RNA was extracted at
`different time intervals after the addition of actinomycin D
`and examined for iNOS mRNA expression by Northern
`blot analysis. Fig. 5 shows that both CMT-3 > doxycycline
`augments iNOS mRNA instability when compared to cells
`incubated with LPS alone. Previous studies have shown that
`murine iNOS mRNA is modulated at post-transcriptional lev-
`els, by TGF|3 [18], Fe(3+) [26] and cycloheximide [27].
`In summary, these experiments demonstrate that CMT-3
`and -8 (both devoid of anti-microbial activity), like doxycy-
`cline and minocycline, can inhibit iNOS activity in murine
`
`macrophages. CMT-3 > CMT-8 at concentrations used in
`this study have been reported to inhibit collagenase activity,
`whereas CMT-5 showed no appreciable effect [28]. Further-
`more, CMT-3 > CMT-8 were found to be more potent in
`their ability to inhibit iNOS expression when compared to
`effects of doxycycline and minocycline as previously reported
`[13]. This study also demonstrates that the mechanism of ac-
`tion of at least two tetracyclines (doxycycline and CMT-3) is
`similar: they render the iNOS mRNA susceptible to degrada-
`tion and thereby decrease iNOS expression and nitric oxide
`production. These experiments support the previous hypoth-
`esis that tetracyclines, independent of their anti-microbial ac-
`tivity, exert pleiotropic functions
`including
`inhibition of
`MMPs, NOS expression, protection against peroxynitrite-de-
`pendent injury, angiogenesis, tumor progression, bone resorp-
`tion and inflammation [10,12,29,30]. We hypothesize that the
`multifunctional properties of tetracyclines may be partly at-
`tributed to their ability to target another pleiotropic signalling
`molecule, NO, that has been shown to exert similar effects on
`many of the pathological conditions and manifestations.
`
`Acknowledgements: We would like to thank CollaGenex Pharmaceut-
`icals, Inc. (Newtown, PA) for support of this study, Dr. James L.
`Lessard (Children's Hospital Medical Center, Cincinnati, OH) for
`the anti-actin antibody, and Ms. Ann Rupel and Susan Forman for
`preparing the manuscript. This work was supported in part by Colla-
`Genex, Inc., Newtown, PA.
`
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