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(Circulation Research. 2001;88:70.)
© 2001 American Heart Association, Inc.

Cellular Biology

Overexpression of 12-Lipoxygenase Causes Cardiac Fibroblast Cell Growth

Yeshao Wen, Jiali Gu, Yaxia Liu, Ping H. Wang, Yanyu Sun, Jerry L. Nadler

From the Division of Endocrinology and Metabolism (Y.W., J.G., J.L.N.), University of Virginia Health Sciences Center (Charlottesville); Department of Diabetes, Endocrinology and Metabolism (Y.L., Y.S.), City of Hope Medical Center, Duarte, Calif; and Department of Medicine and Biological Chemistry (P.H.W.), University of California (Irvine).

Correspondence to Jerry L. Nadler, MD, Division of Endocrinology and Metabolism, University of Virginia Health Sciences Center, MR4 Building, Room 5150, Lane Road, Charlottesville, VA 22908. E-mail jln2n{at}virginia.edu

	Abstract

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Abstract—Evidence suggests that leukocyte type 12-lipoxygenase (12-LO) plays an important role in cell growth. However, the role of 12-LO in cardiac cell growth has not been tested. We have now stably overexpressed 12-LO cDNA in rat fetal cardiac fibroblasts to evaluate the role of the 12-LO pathway in cardiac cell growth. Overexpression of 12-LO increased cell [³H]leucine incorporation by 2.1±0.1-fold (P<0.01) and cell protein content by 2.2±0.3-fold (P<0.01) over mock-transfected cells. These findings were confirmed in additional clones. Baicalein, a 12-LO enzyme inhibitor, dose-dependently inhibited serum-induced leucine incorporation in cardiac fibroblast cells as well as partially inhibited leucine incorporation in cells overexpressing 12-LO. 12-LO overexpression also caused cell [³H]thymidine incorporation to increase by 3.4±0.3-fold (P<0.01). Cell flow cytometry analysis showed that the size of 12-LO–overexpressing cells was markedly enlarged compared with that of mock-transfected cells. The fibronectin content of the 12-LO–overexpressing cardiac fibroblasts was also significantly increased. We next evaluated the effects of 12-LO RNA overexpression on kinase pathways linked to cellular growth. The overexpression of 12-LO enhanced extracellular signal-regulated kinase activity (4.1±0.5-fold), c-Jun NH₂-terminal kinase activity (2.9±0.5-fold), and p38 mitogen-activated protein kinase activity (2.2±0.3-fold). Pretreatment with SB202190 (100 nmol/L), a specific inhibitor of p38, prevented the increases in protein content of 12-LO–overexpressing cardiac fibroblast cells. These data clearly demonstrate that the overexpression of 12-LO causes cell growth of cardiac fibroblasts, thus supporting the role of 12-LO as a novel growth-promoting pathway in the heart.

Key Words: lipoxygenase • HETE • cell proliferation • cells

	Introduction

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Cardiac hypertrophy is an adaptational state to prior hypertension and is a major risk factor associated with heart failure. Cardiac hypertrophy is indexed by increases in thickness of the left ventricle wall, heart weight, and heart weight–to–body weight ratio. From a morphological point of view, there are 2 structural changes that occur in the hypertrophied myocardium in hypertension: (1) cardiomyocyte growth as reflected by an increase in cell breadth and/or length, which creates an increment in the mass and thickness of the myocardium; and (2) cardiac fibroblast growth as expressed as structural remodeling of the interstitium. In left ventricular hypertrophy that accompanies hypertension, the extracellular space is frequently the site of the abnormal accumulation of fibrillar collagen and fibronectin. This interstitial and perivascular fibrosis accounts for abnormal myocardial stiffness and ultimately ventricular dysfunction.¹

Cardiac fibrosis is characterized by fibroblast growth and the concomitant deposition of extracellular matrix. Although the relationship between cardiac fibrosis and angiotensin II (Ang II) has been known for many years, direct interactions of Ang II with the cardiac fibroblast and myocyte have been described only recently. The presence of Ang II receptors on neonatal and adult rat cardiac fibroblasts is now established.² These receptors, predominantly of the Ang II type 1 (AT₁), lead to growth, increases in protein synthesis, and induction of extracellular matrix protein gene expression. However, the mechanism involved in Ang II and other growth factor actions is far from clarified.

Evidence suggests that the leukocyte type 12-lipoxygenase (12-LO) enzyme plays an important role in cell growth and that its product derived from arachidonic acid, 12-hydroxyeicosatetraenoic acid (12-HETE), is a potent cell growth–promoting agent. This action of 12-HETE was shown in Chinese hamster ovary cells stably overexpressing the AT_1a receptors (CHO-AT_1a).³ 12-HETE also has been shown to lead to the hypertrophy of vascular smooth muscle cells.⁴ 12-HETE has been demonstrated to be an activator of extracellular signal-regulated protein kinase (ERK),³ c-Jun N-terminal kinase (JNK),⁵ and p21-activated kinase (PAK)⁶ in this cell line.

The role of the 12-LO pathway in cardiac growth has not been previously evaluated. To study the effect of 12-LO in cardiac cellular growth, we stably overexpressed mouse leukocyte type 12-LO cDNA in cardiac fibroblast cells derived from fetal rats. Our results demonstrate that the overexpression of 12-LO increases DNA, RNA, and protein synthesis. Furthermore, the overexpression of 12-LO activates mitogen-activated protein (MAP) kinases and leads to increases in extracellular matrix production. These results support a role of 12-LO as a novel growth-promoting pathway in the heart.

	Materials and Methods

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Reagents
DMEM and FBS were supplied by Irvine Scientific. BSA (fatty acid free), myelin basic protein, ADP-ribosyltransferase C₃, leupeptin, and aprotinin were obtained from Sigma Chemical Co. 12-HETE was from BIOMOL. SB202190 was from Upstate Biotechnology Inc. GST-c-Jun amino acids 1 to 79 plasmid was kindly provided by Dr Michael Karin (University of California San Diego). ATF-2, JNK₁, PAK₁, and p38 MAP kinase antibodies were from Santa Cruz Biotechnology Inc. [-³²P]ATP, [³H]thymidine, [³H]leucine, and [³H]uridine were from New England Nuclear Corp. PUR vectors and. puromycin were from Clontech. The calcium phosphate kit was purchased from Pharmacia. The pcDNA1/ML12-LO vector was kindly provided by Dr C.D. Funk (University of Pennsylvania, Philadelphia, Pa).

Cell Culture and 12-LO Stable Transfection
Cardiac fibroblast cells from fetal rat heart were provided by Dr Ping H. Wang and colleagues at the University of California Irvine.⁷ Mouse leukocyte type 12-LO cDNA was stably transfected into cardiac fibroblast cells (ML-12-LO cells) according to the calcium phosphate DNA precipitation method. Cardiac fibroblast cells were maintained in culture in DMEM with 10% FBS containing 20 mmol/L HEPES, pH 7.4, penicillin, and streptomycin at 37°C in 5% CO₂ and 95% air. To generate ML12-LO cells, cardiac fibroblast cells were seeded at density of 1x10⁶ cells in a 100-mm dish. pcDNA1/ML12-LO vector and pPUR vector, a plasmid conferring resistance to puromycin, were cotransfected with the calcium phosphate DNA precipitation method according to the manufacturer’s instructions (Pharmacia). The 12-LO–overexpressing cell line is known as M4.7 cells. The empty vector pcDNA 1 (Invitrogen) without the insert (12-LO cDNA) was used as the control. This cell line is known as P3 cells. Vectors were purified with an Endofree plasmid kit (Qiagen); 48 hours later, the transfected cells were split 1:15. Selection was then initiated with 2 µg/mL puromycin in the cultures to select cells that expresses resistance to this marker. Individual resistant clones were isolated 2 to 3 weeks later and expanded into cell lines. Transfected cells were maintained in the medium containing 10% FBS and 2 µg/mL puromycin.

Immunoblotting
To analyze the expression of the 12-LO protein, immunoblotting was performed as previously described.⁸ The polyclonal antibody against amino acids 646 to 662 peptide sequence of porcine leukocyte 12-LO protein was raised in rabbits. The antibody showed excellent cross-reactivity with murine leukocyte 12-LO. Cells were lysed in lysis buffer containing PBS (pH 7.4), 1% Triton X-100, 0.1% SDS, and standard protease inhibitor cocktail. Lysates were centrifuged, and the supernatants were collected for assays; 20 µg of the protein was resolved through 10% SDS-PAGE and subsequently transferred to a polyvinylidine difluoride membrane. After the membrane was incubated overnight in blocking buffer (Tropix Inc), 12-LO antibody was added at a 1:1000 dilution. Next, an alkaline phosphatase–coupled goat anti-rabbit secondary antibody was added at a 1:10 000 dilution. The protein bands are visualized with chemiluminescence substrate and the Western Light chemiluminescent detection system (Tropix Inc).

Measurement of the 12-LO Product, 12-HETE
12-HETE was extracted from the supernatant with the use of C18 Bond Elut Columns as previously described.⁹ The cell pellets were first deacylated to release the cell-associated HETEs.⁹ The LO products in the supernatants as well as extracts were quantified with a specific radioimmunoassay. The specific antiserum for 12-S-HETE was obtained from Perspective Biosystems, and authentic ³H-label tracers were purchased from NEN Research Products. The unlabeled 12-S-HETE standard was obtained from BIOMOL Research Laboratories. Separation of bound and free labeled HETE was achieved with the use of dextran-coated charcoal. The sensitivity of the assay is 10 pg/mL with an intra-assay variation of 8%.

Determination of [³H]Thymidine, [³H]Leucine, or [³H]Uridine Incorporation and Protein Content
Cardiac fibroblast cells were grown on 6-well culture plates for 3 days. At 20 to 24 hours after depletion with depletion buffer (DMEM containing 20 mmol/L HEPES, pH 7.4, 0.2% BSA, 0.4% FBS), cells were continuously cultured in the depletion medium that contained 1 µCi/mL ³H-isotope.¹⁰ At 20 hours later, the medium was aspirated and cells were rapidly washed twice with 1 mL cold PBS solution and once with 1 mL 10% trichloroacetic acid (TCA) and incubated in 1 mL fresh 10% TCA at 4°C for 30 minutes. The TCA-insoluble material was washed twice with 95% ethanol, and fixed cellular material was solubilized in 0.1N NaOH at 24°C for 2 hours. Sample was divided into 6 wells. Three wells were used for incorporation and protein content measurements, and 3 wells were used for cell counting. The ³H-isotope incorporation was determined with liquid scintillation spectrometry. The protein content was determined according to the Bradford method. Cells were counted with a Coulter counter. The data were normalized as cpm/10⁶ cells or µg protein/10⁶ cells and finally expressed as the fold over mock condition.

Flow Cytometry
Cell DNA was stained with propidium iodide and analyzed at the Analytical Flow Cytometry Facility at the City of Hope National Medical Center.

Soluble and 1% Deoxycholate–Insoluble Fibronectin Measurement
Confluent (80% to 90%) mock-transfected (empty vector–transfected) cells or 12-LO–transfected cells were depleted with depletion medium for 24 hours. The supernatants were assayed for released fibronectin, and washed cell layers were extracted with 1% deoxycholate.¹¹ The fibronectin in supernatants was regarded as soluble fibronectin, whereas the 1% deoxycholate–insoluble material was taken as a fibrillar form of fibronectin. Fibronectin in all samples was determined with a double-antibody sandwich ELISA with the methods provided by the manufacturer (DACO Corp). A polyclonal rabbit anti-human fibronectin (1:1000) was used as the coating antibody, and the detection antibody was a peroxidase-conjugated rabbit anti-human fibronectin (1:2000).

MAP Kinase Activity Measurement
ERK1/2, JNK-1, and p38 activities were measured with the immune complex kinase assay method as previously described.^{3 5 12} Cells were growth arrested through incubation in depletion DMEM for 24 hours. After being washed twice with cold PBS, the cells were lysed with lysis buffer consisting of 50 mmol/L HEPES, pH 7.5, 150 mmol/L NaCl, 5 mmol/L MgCl₂, 5 mmol/L EGTA, 50 mmol/L NaF, 10 mmol/L Na pyrophosphate, 2.5% glycerol, 1% Nonidet P-40, and 1 mmol/L Na₃VO₄ with the protease inhibitors, including PMSF, leupeptin, and aprotinin. The lysate was centrifuged at 14 000g at 4°C for 10 minutes. Protein determination of lysate was made according to the Bradford method. For immunoprecipitation, 50 µg of lysate protein was incubated with JNK, p38, and ERK antibodies in the lysis buffer; the mixture was rotated at 4°C overnight; and then the solution was added to 60 µL protein A–Sepharose. After a 1-hour incubation at 4°C, the beads were washed 4 times with diluted lysis buffer, and the pelleted beads were resuspended in 60 µL kinase buffer containing 2 µg GST-c-Jun (amino acids 1 to 79) for JNK assay, 2 µg ATF-2 for p38 assay, or 2 µg myelin basic protein for ERK assay and 20 µmol/L ATP and 5 µCi [-³²P]ATP. After 30 minutes at 30°C, the reaction was stopped with 5[times] Laemmli’s sample buffer and resolved on 12% SDS–polyacrylamide gel, followed by autoradiography.

Data Analyses
The results are expressed as mean±SEM from combined experiments as noted in each legend. ANOVAs with Dunnett’s or Tukey-Kramer multiple comparison tests or Student’s t tests were used to analyze the data. Autoradiograms of the JNK activity studies were analyzed with an automated computerized densitometer (SCISCAN 5000; US Biochemical). Measurements were made in the linear range, and the values are expressed as arbitrary optical density units or fold over control.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Characterization of Cardiac Fibroblast Cells Stably Overexpressing 12-LO
Cardiac fibroblast cells were cotransfected with the 12-LO cDNA expression vector and pPUR-resistant vector. Cells were screened under the puromycin-selective medium. Several surviving clones were isolated and expanded to independent clones for further characterization. To characterize cell lines that stably overexpress 12-LO at different levels, 12-LO immunoblotting assay and activity assays were performed. Figure 1 shows 12-LO protein expression in ML12-LO–overexpressing clones (M4.7 and M19) and the control (empty vector) clone (P3). It is clear that M4.7 and M19 clones express much higher steady-state levels of the 12-LO protein than does mock clone P3 (Figure 1). M4.7 clone expresses a greater amount of 12-LO protein than M19. 12-LO protein was only slightly detectable in clone P3. 12-LO activity illustrated by the level of its product, 12-S-HETE, released into cultured medium from clone M4.7 as well as control clone P3 was measured with a radioimmunoassay. 12-S-HETE release from cardiac fibroblasts stably overexpressing 12-LO was 4-fold greater than that for the control clone P3 (4.3±0.2-fold over control, n=3, P<0.01).

View larger version (16K): [in this window] [in a new window]   Figure 1. Western blot of 12-LO protein expression in mock-transfected cells and in cells overexpressing 12-LO. A polyclonal antibody with 1:1000 dilution against amino acids 646 to 662 of leukocyte 12-LO was used. Then an alkaline phosphatase coupled with goat anti-rabbit second antibody was added at 1:10 000 dilution. The protein bands were visualized by chemiluminescence substrate and Western Light chemiluminescent detection kit (Tropix Inc). Clones M4.7 and M19 were transfected with mouse leukocyte 12-LO plasmid. P3 represents the clone transfected with the empty vector plasmid.

Effect of Leukocyte 12-LO Overexpression on Cell Growth
To explore the effect of 12-LO overexpression on cell growth, clones of M4.7 and P3 were labeled with [³H]thymidine to evaluate DNA synthesis or with [³H]leucine as an indication of protein synthesis. Figure 2 illustrates that the overexpression of 12-LO increased leucine incorporation by 2-fold (2.1±0.1-fold, n=4 in triplicate, P<0.01) over control cells (clone P3), whereas thymidine incorporation was increased by 3-fold (3.4±0.3-fold, n=4 in triplicate, P<0.01) in 12-LO–overexpressing cells over that in control cells. [³H]Uridine incorporation in 12-LO–transfected cells was >5-fold greater than that seen in control cells (5.6±0.7-fold, n=3, P<0.01). Finally, protein content in these 2 cell lines was measured according to the Bradford method. The protein content in cardiac fibroblasts overexpressing 12-LO was 2.2±0.3-fold greater that in control cells when expressed as micrograms per 10⁶ cells (P<0.01, n=5). To analyze the role of 12-LO in leucine incorporation of cardiac fibroblast cells, we studied 2 additional clones of mock-transfected cells, P2 and P4, and 3 additional clones of cells overexpressing 12-LO, M19, M1.5, and M1.6. The comparison of their leucine incorporation shown in Figure 3 shows that the leucine incorporation in these 3 lines of cells overexpressing 12-LO is significantly increased compared with that in mock-transfected cells, suggesting that these 3 clones of cells overexpressing 12-LO are similar to M4.7 cells. Furthermore, baicalein, an inhibitor of 12-LO, was able to inhibit 0.4% serum-induced increases in leucine incorporation dose-dependently in cardiac fibroblasts. The leucine incorporation was 77.2±0.016% of that in control cells (n=3, P<0.001) when baicalein was at 1 µmol/L,. The leucine incorporation was reduced to 27.4±0.01% of that in control cells when baicalein was at 10 µmol/L (n=3, P<0.001). Baicalein was then used to test whether the 12-LO overexpression–induced increases in protein content could be inhibited. At 1 µmol/L, baicalein had little effect on leucine incorporation in empty vector–expressing cells, but, in contrast, 1 µmol/L baicalein inhibited 50% of leucine incorporation seen in the 12-LO–overexpressing cells (Figure 4). These results suggest a specific effect of the 12-LO pathway in leading to protein content increases in cardiac fibroblast cells. 12-LO overexpression also decreased the cell split rate compared with the increase in cell number of mock-transfected cells. When the same number of cells were seeded and grown in 10% serum for 3 days, the total cell number of both cell lines was increased; however, the cell number of control cells was 2-fold greater than that seen in 12-LO–transfected cells (2.3±0.4-fold over 12-LO–transfected cells, P<0.01, n=5). The cell number of 3 new clones of cells was also about the half of cell number of 2 new clones of mock-transfected cells after 3 days of culture under the same condition.

View larger version (15K): [in this window] [in a new window]   Figure 2. Effect of leukocyte 12-LO overexpression on cardiac fibroblast cell growth. Fibroblast cells were grown on 6-well plates with 10% FBS for 3 days. Cells were depleted with DMEM containing 20 mmol/L HEPES, pH 7.4, 0.2% BSA, and 0.4% FBS. After depletion for 20 to 24 hours, cells were continuously cultured in the depletion medium containing 1 µCi/mL [3H]thymidine or [3H]leucine. At 20 to 24 hours later, cells were washed and treated for determination of 3H-isotope incorporation as described in Materials and Methods.

View larger version (30K): [in this window] [in a new window]   Figure 3. Different clones of cells overexpressing 12-LO increase leucine incorporation. Different clones of cells were grown on 6-well plates and depleted under the same conditions as for Figure 2. After depletion for 24 hours, cells were continuously cultured in the depletion medium containing 1 µCi/mL [3H]leucine. At 20 to 24 hours later, cells were washed and treated for determination of 3H-isotope incorporation as described in Materials and Methods. Each point is an average (mean±SEM) from 3 separate samples. Identical results were seen in 2 experiments.

View larger version (37K): [in this window] [in a new window]   Figure 4. Effect of 12-LO inhibitor baicalein on leucine incorporation of 12-LO–overexpressing cardiac fibroblast cells. Fibroblast cells expressing empty vector or 12-LO were grown on 6-well plates and depleted under the same conditions as for Figure 2 but with 1 or 10 µmol/L baicalein or ethanol in the control. At 20 to 24 hours later, cells were continuously cultured in the depletion medium containing 1 µCi/mL [3H]leucine. At 20 to 24 hours later, cells were then washed and treated for determination of 3H-isotope incorporation as described in Materials and Methods. Each point is an average (mean±SEM) from 3 separate samples. Identical results were seen in 2 experiments.

Flow cytometry was used to evaluate cell size. Figure 5 (left) illustrates the forward scatter seen from the histogram of control and 12-LO–transfected cells. Overlapping of forward scatter was redrawn with the use of a computer program (Figure 5, right). The size of the majority of the 12-LO–overexpressing cells was shifted to the right compared with that of control cells. The cell morphology was examined with hematoxylin and eosin (H&E) staining. Figure 6 shows that the sizes of 12-LO–transfected cells were markedly larger than those of control cells, confirming the data from flow cytometry. Figure 6 also illustrates that the size of the nuclei in 12-LO–transfected cells was much larger than that of the nuclei in control cells. Twenty nuclei in Figure 6 were arbitrarily chosen, and the long axis and numbers of the nucleoli were measured. The mean long axis of nuclei of 12-LO–transfected cells was 6.4±0.21 mm compared with 4.15±0.26 mm in control cells, indicating that the mean long axis of nuclei in 12-LO–transfected cells was 1.54-fold greater than that in control cells (P<0.001). The analysis also demonstrated that the mean number of nucleoli was 2.95 in 12-LO–transfected cells versus 1.5 in mock-transfected cells (P<0.001). These results suggest that 12-LO overexpression leads to morphological changes in rat cardiac fibroblasts.

View larger version (17K): [in this window] [in a new window]   Figure 5. Overexpression of 12-LO increases cardiac fibroblast cell size measured with flow cytometry. Left, Forward scatter histogram of the flow cytometry analysis screen: 106 mock-transfected cells (top) and 106 cells overexpressing 12-LO were used. Right, Overlapping of 2 forward scatters in the left panel.

View larger version (100K): [in this window] [in a new window]   Figure 6. Morphology of mock-transfected cells (top) and cells overexpressing 12-LO (bottom). Cells (1x105) were cultured on a chamber slide and incubated at 37°C for 24 hours. After washing with PBS, the cells were fixed in 100% acetone and then stained with H&E: hematoxylin for 5 minutes and eosin for 1 minute.

Overexpression of 12-LO Stimulates MAP Kinase
To evaluate the effects of overexpression of 12-LO on MAP kinase activity, ERK, JNK, and p38 activities were measured in control cells and in cells overexpressing 12-LO. ERK, JNK, and p38 activities in 12-LO–transfected cells were markedly increased compared with those in control cells. The overexpression of 12-LO stimulated ERK by 4-fold (4.1±0.5-fold, n=3, P<0.01), p38 by 2-fold (2.2±0.3-fold, n=3, P<0.02), and JNK by 3-fold (2.9±0.5-fold, n=3, P<0.02).

Effect of MAP Kinases on Cell Growth in 12-LO–Transfected Cells
To elucidate the role of p38 and ERK activation in fibroblast cell hypertrophy, SB202190, a specific inhibitor of p38 MAP kinase, or PD 58059, a specific MEK inhibitor, was used. Figure 7 illustrates that the pretreatment of 12-LO–overexpressing cardiac fibroblasts with 100 nmol/L SB202190 compound for 24 hours prevented the increases in protein content seen in these cells. Pretreatment with SB202190 did not significantly alter the cell protein content in mock-transfected cells, implying that p38 MAP kinase activation may be important for the 12-LO–induced protein content increase. In contrast, the compound PD58059, an inhibitor of MEK, had no effect on leucine incorporation in 12-LO– or mock-transfected cells (data not shown).

View larger version (17K): [in this window] [in a new window]   Figure 7. Compound SB202190 prevents the increase in cell protein content in 12-LO–overexpressing cells. Cells were grown on 6-well plates and depleted as described in the legend for Figure 2. After depletion for 24 hours, cells were treated with compound SB202190 in the same depletion medium for 24 hours, and then protein content was determined according to the Bradford method.

Overexpression of 12-LO Increases Matrix Protein Content
Increasing evidence suggests that extracellular matrix (ECM) proteins play a key role in cell adhesion, migration, and growth. To evaluate the effect of 12-LO overexpression on matrix production, fibronectin protein content in mock- and 12-LO–transfected cells was measured with a specific ELISA. Figure 8 (left) illustrates that 12-LO–transfected cells released 3.7-fold more fibronectin than did mock-transfected cells (P<0.01). To measure the fibrillar form of fibronectin, a serial extraction method was used. 12-LO–transfected cells contained 3.4-fold more of the extracellular fibrillar form of fibronectin than did control cells (P<0.01) (Figure 8, right).

View larger version (14K): [in this window] [in a new window]   Figure 8. Overexpression of 12-LO stimulates the formation of soluble and fibrillar forms of fibronectin. Cells were grown on 100-mm dishes and then depleted as described in the legend for Figure 2. After depletion for 20 to 24 hours, the supernatant was taken for soluble fibronectin measurement. The remaining cells were washed twice with cold PBS and then solubilized with 1% deoxycholate buffer. The 1% deoxycholate–insolubilized pellet was taken for measurement of the fibrillar form of fibronectin. Fibronectin normalized per 106 cells was determined with a specific ELISA.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have demonstrated for the first time that stable overexpression of the leukocyte type 12-LO causes cell growth and matrix production in fetal rat cardiac fibroblasts. It was clearly shown that cell lines M4.7 and M19 transfected with 12-LO cDNA have greater 12-LO protein expression and activity than the mock-transfected line P3. The faint protein band in the P3 cells in Figure 1 represents endogenous 12-LO expression in parent cardiac fibroblast cells. These data that show increases in the specific 12-LO product 12-S-HETE suggest the 12-LO protein in M4.7 cells is functionally intact.

The results indicate that overexpression of 12-LO increases DNA, RNA, and protein synthesis. To analyze the role of the 12-LO pathway in cell growth of cardiac fibroblast cells, 2 additional clones of mock-transfected cells and 3 additional clones of cells overexpressing 12-LO were selected, and their leucine incorporation was compared. That all 4 clones of cells overexpressing 12-LO display significant increases in leucine incorporation compared with 3 clones of mock-transfected cells strongly suggests the specific role of the 12-LO pathway the protein content increase of cardiac fibroblasts. Furthermore, our results that show baicalein, a specific inhibitor of 12-LO, was able to inhibit 0.4% serum-induced leucine incorporation dose-dependently with 72% reduction when the concentration of baicalein was 10 µmol/L and that 1 µmol/L baicalein inhibited 50% of 12-LO overexpression–induced leucine incorporation suggest that 12-LO does play an important role in the protein content increase of cardiac fibroblasts. Interestingly, we observed in the current study that overexpression of 12-LO increased cell size with a decrease in cell split rate, supporting hypertrophy rather than hyperplastic changes in these cells. The decrease in the cell split rate is unlikely to be due to 12-LO overexpression–induced toxicity or apoptosis, because there were no cells floating nor cells rounding up that could be seen. In addition, the cells retained their normal shape and fully attached in the presence of 0.4% FBS. We propose that cells overexpressing 12-LO retain characteristics of fibroblasts, yet they have been partially conferred with some features of myocytes that have the function of cell hypertrophy and can account for the increase in protein content per 10⁶ cells and cell size. The evidence to support the hypothesis that cardiac fibroblasts overexpressing 12-LO show characteristics of cell hypertrophy comes from the following observations. (1) The nucleus size and nucleolus number were markedly increased in response to the overexpression of 12-LO compared with mock-transfected cells. A study has shown that changes in the size of the nucleus and in the numbers of nucleoli are a sensitive indication of early hypertrophy of the myocardium in humans.¹³ 2. The increase in fibronectin released into the medium as well as in the formation of the fibrillar form of fibronectin also supports the growth effect of 12-LO. More studies are needed to determine the precise mechanism of these changes in the fibroblasts, because cells can enlarge and the nucleus can change shape under circumstances other than hypertrophy.

We next examined the mechanism by which overexpression of 12-LO facilitated cardiac fibroblast cell growth. The data suggest that p38 MAP kinase could play a role in 12-LO–induced protein content increase, because overexpression of 12-LO significantly enhanced p38 MAP kinase activity. Moreover, treatment of 12-LO–transfected cells with SB202190, a pyridinyl imidazole compound that specifically inhibits p38 kinase activity,^{14 15} led to a reduction in leucine incorporation. These results are consistent with data in myocytes showing that p38 MAP kinase activity is increased in hearts after chronic transverse aortic constriction, coincident with the onset of ventricular hypertrophy.¹⁶ Additional evidence for the role of p38 kinase in cardiac growth is that adenoviruses expressing activated MKK3 or MKK6 increase p38 activation and lead to hypertrophy of cardiac myocytes, promotion of sarcomeric organization, and increases in ANF expression. In addition, adenoviruses expressing dominant negative mutants or a specific inhibitor for the p38 kinase partially block the Ras-induced cardiac cell hypertrophy.¹⁷

To date, at least 4 isoforms of p38 MAP kinases have been identified, and it is likely that these isoforms will have differential actions.^{14 18 19} Two well characterized isoforms, and ß, share extensive sequence homology and a broad range of tissue distribution, including relatively high levels in the heart.^{14 18} Recent data have shown that the activation of p38ß can induce several features of the hypertrophic response, whereas the activation of p38 antagonizes these effects and results in cell death.¹⁶ In future studies, it will be of interest to characterize which isoforms of p38 kinase are induced by 12-LO overexpression in cardiac fibroblast cells.

Overexpression of 12-LO also markedly increased JNK activity. The direct hypertrophic effect of JNK activation in cardiac myocytes has been demonstrated by overexpressing a specific upstream activator, MKK7, using recombinant adenovirus-mediated gene transfer.²⁰ However, the precise role of JNK activation in cardiac fibroblast cell growth has not yet been tested. ERK also plays a pivotal role in signal cascades leading to cell growth.²¹ There is evidence to show that depletion of ERK using an antisense oligodeoxynucleotide approach downregulates the transcriptional and morphological changes of hypertrophy induced by phenylephrine.^{22 23} However, ERK inhibition could not suppress Ang II–induced organization of actin filament in myocytes.²³ Furthermore, ERK inhibition did not suppress Ang II–induced overall protein content increases in cardiac myocytes.²⁴ Therefore, the role of ERK activation in cardiac cell growth is still unclear. In the present study, we observed that the overexpression of 12-LO increases ERK activity; however, the compound PD58059, an inhibitor of MEK, had no effect on leucine incorporation in 12-LO–transfected cardiac fibroblasts, suggesting that ERK is not involved in 12-LO–induced protein synthesis. Further experiments will be needed to understand the function of ERK activation in 12-LO overexpression–induced cardiac fibroblast cell growth.

Evidence suggests that cell growth parallels the accumulation of surface fibronectin matrix and that removal of the matrix prevents growth.²⁵ Similarly, inhibition of fibronectin matrix assembly in various ways inhibits cell growth.²⁶ Increased mRNA and protein levels of fibronectin have been observed in ischemia, triiodothyronine treatment, or mineralocorticoid-induced hypertensive hearts,²⁷ as well as in spontaneously hypertensive hearts.²⁸ Although available data suggest that fibronectin may have a causative role in cardiac hypertrophy, a functional role for fibronectin in the hypertrophic heart has not been definitively established. Our data that overexpression of 12-LO facilitates cardiac fibroblast protein content increase as well as fibronectin expression clearly implicate the 12-LO pathway as a potentially important component that integrates cell protein content increase and extracellular matrix protein expression. In particular, the results show that the overexpression of 12-LO in cardiac fibroblast cells not only causes increases in fibronectin released into medium but also markedly increases 1% deoxycholate–insolubilized fibronectin, the extracellular fibrillar form of fibronectin.

These results support the role of 12-LO activation as a potentially important mechanism that leads to protein content increases in cardiac fibroblasts through the activation of growth-related kinases and matrix formation.

	Acknowledgments

 
This study was supported by American Heart Association, Greater Los Angeles Affiliate, Scientist Development Grant 1180-SI1 (Dr Wen) and National Institutes of Health grants R01-DK-39721 and P01-HL-55798 HBL1 (Dr Nadler). The authors would like to thank L. Lanting for the 12-LO activity assay, N. Gonzales for preparation of the figures, and A. Fontanilla for preparation of the manuscript.

	Footnotes

 
Original received September 22, 2000; revision received October 27, 2000; accepted October 27, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
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