[Frontiers in Bioscience 3, a16-22, February 15, 1998]

	[Frontiers in Bioscience 3, a16-22, February 15, 1998] Reprints PubMed CAVEAT LECTOR
	EFFECT OF TGF-BETA 1 ON PDGF RECEPTORS EXPRESSION IN HUMAN SCAR FIBROBLASTS Messadi DV^1,2 , Le A¹, Berg S¹, Huang G¹, Zhuang W¹ and Bertolami CN³ ¹School of Dentistry, ²Dental Research Institute, University of California, Los Angeles, California, ³School of Dentistry,University of California, San Francisco, California, USA Received 1/8/98 Accepted 2/3/98 3. MATERIALS AND METHODS The effect of TGF- beta 1 treatment on PDGF alpha and beta receptor cell surface expression for NSk, NSc and keloid fibroblasts was analyzed using RadioImmunoBinding assay. All cells were serum starved for 24 hours; one half of the cultures were then preincubated for 24 hours with 10 ng/mL of TGF- beta 1. Using monoclonal antibodies specific for PDGF alpha and beta receptors, the TGF- beta 1 stimulatory effect on the different cell lines was examined. 3.1 Cell cultures Three replicate cultures each of human NSk, NSc and keloid fibroblasts were cultured as previously described (14). Patients ages ranged from 18 to 50 years. Normal skin tissue samples were matched for age, race and sex. In brief, freshly biopsied tissues, were obtained from the operating room of the University of California Medical Center, Los Angeles (UCLA) and Martin Luther King Hospital, Los Angeles as part of therapeutic procedures. Tissues were enzyme digested, the cells being propagated and maintained in Dulbecco’s Modified Eagle’s Medium (D-MEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin streptomycin. The cells were studied at passages 4-8. 3.2 Addition of exogenous TGF- beta 1 Twenty-four hours before treating cultures with TGF-beta1, regular medium was replaced with serum-free medium. Half of the preconfluent cultures received TGF-beta1 (Sigma Company, St. Louis, MO) at a concentration of 10 ng/mL in serum-free medium for 24 hours. The TGF-beta1-containing medium was replaced with serum-free medium for another 24 hours before harvesting. This dosage and exposure regimen is based on our previous work (13) and the work of Westergren-Thorsson and coworkers (15) showing that TGF-beta1-induced effects on human fibroblast proliferation and activation lasts up to 48 hours after initial exposure. 3.3 Radioimmunobinding assay Quantitative analysis was performed to determine whether statistically significant differences exist in the expression of PDGF alpha and beta receptors in response to the addition of exogenous TGF-beta1 for the different cell lines. A Dot Microfold apparatus from V&P Scientific, Inc. (San Diego, CA) was used. The different cell lines were fixed with 2% glutaraldehyde and seeded at 5 x 104 cells in the manifold. The manifold has the configuration of a 96 well plate whose bottom surface consists of glass microfilter paper (Whatman, Clifton, NJ). Each well was incubated with 100 ml of the various primary antibodies—either anti-PDGF alpha receptor antibody, PDGF-beta receptor antibody (R&D System Inc., Minneapolis, MN) or mouse IgG as a negative control. The wells were washed several times with PBS+ 2% FBS. Radiolabeled secondary antibody [125I]-conjugated mouse antibody was used at 0.1 mCi/well (ICN, Irvine CA), vacuum applied, and vigorous washing performed to eliminate any nonspecific binding. Wells were cut separately and each was counted for its immune reactivity using a liquid scintillation counter (14). Results were expressed as disintegration per minute (dpm) for each cellular condition and each antibody tested. 3.4 RNA extraction To determine mRNA levels for PDGF-alpha and beta receptors, fibroblasts from the various tissues were cultured in 75-cm² tissue culture flasks in the presence or absence of TGF-beta1 as described above. At the end of the incubation period, total cellular RNA was extracted from cell cultures using an acid guanidium thiocyanate-phenol-chloroform described by Chomczymski and Saachi (16). Briefly, cells were washed twice with PBS, lysed by adding 10 mL of 4M guanidium thiocyanate buffer, and collected in 50 ml conical tubes. The following solutions were added in succession with vortexing 2-5 minutes each time: 2M sodium acetate pH 4, followed by water saturated phenol, followed by chloroform-isoamyl alcohol. The mixture was centrifuged at 4oC, RNA was ethanol precipitated, centrifuged and resuspended in lysing buffer, centrifuged and the RNA pellet resuspended in 75% ethanol at room temperature. The RNA mixture was then centrifuged, vacuum dried, and the RNA pellet dissolved in 0.5% SDS. Absorbance was checked at OD 260. 3.5 Polymerase chain reaction amplification Total RNA was isolated from AG 1518 human foreskin fibroblasts (Human Genetic Mutant Cell Repository, Camden, New Jersey) by the method of Chirgwin and coworkers (17). This was used for first strand synthesis in accordance with the work of Claesson-Welch and coworkers (18) who have shown abundant amplification product for PDGF-alpha receptor by this method. RT-PCR was performed using the GeneAmp RNA PCR kit (Perkin Elmer Cetus, Norwalk, CT) according to the manufacturer’s instructions. For cDNA amplification, PCR was performed for 30 cycles. Throughout successive stages of re-amplification and purification, 1.6% Agarose Gels (1XTBE, ethidium bromide) was used to monitor the integrity of the product. The presence of a PDGF-alpha receptor transcript was indicated by the PCR amplification of appropriately sized product generated by the deoxyoligonucleotide primers derived from the published PDGF-alpha cDNA sequences (18) shown to yield a PCR product of 989 bp. PDGF-beta receptor and beta-Actin cDNA probes, the latter used as positive control, were purchased from American Type Culture Collection (Rockville, MD). 3.6 Northern blot analysis Ten mg of total RNA was separated by electrophoresis in a 0.66 M formaldehyde/1 % agarose gel according to the method of Sambrook and coworkers (19) using a 0.24 to 9.5 kb RNA ladder (BRL, Life Technologies, Inc., Gaithersburg, MD) as size markers. Gels were stained for 10 minutes in ethidium bromide and destained in water. Bands were visible under UV light and then photographed. The agarose gels were transferred to Zeta probe blotting membranes (Bio-Rad Laboratories, Richmond, CA). The membranes were cross- linked by ultraviolet irradiation using a Stratalinker (Stratagene, La Jolla, CA), then prehybridized in the prehybridization buffer containing 50% deionized formamide, 5X Denhardt’s solution, 1% sodium dodecyl sulfate (SDS), 5X SSPE and 100 mg/ml herring sperm DNA. Membranes were prehybridized for 6 hours at 42oC, then hybridized overnight at 42oC with the different probes which were labeled using a random primed labeling kit (Boehringer Mannheim Biochemicals, Indianapolis, IN) according to the manufacturer’s protocol with [alpha-32P] dCTP (-3000 Ci/mmol, Amersham Corporation, Arlington Heights, IL.) in the same prehybridization buffer but without the herring sperm DNA. Membranes were washed twice in 2X SSPE for 15 minutes at room temperature, twice in 2 X SSPE/2% SDS for 45 minutes at 55oC and twice in 0.1 x SSPE for 15 minutes at room temperature. Then membranes were exposed to Kodak X Omat AR film at –70oC overnight. 3.7 Western blot analysis Confluent fibroblast cultures derived from NSk, NSc and keloid in the presence or absence of TGF-beta1 as described above, were solubilized in cold lysis buffer containing 10mM Tris-HCl, 150 mM NaCl, (TSA) 5 mM EDTA, 1.2% Triton X-100, 2 mM phenyl methylsulfonyl fluoride (PMSF), 2 mM iodoacetamide, 0.15 U/ml aprotinin, and 10 ug/ml leupeptin. Cell suspensions were sheared using a syringe and centrifuged at 2000 rpm for 5 minutes at 4^oC. Lysate pellets were stored at –70^o C. The protein concentration of the different cell lysates were measured using the BCA protein assay kit (Pierce, Rockford, IL) and analyzed by a spectrophotometer. Immunoprecipitation of PDGF alpha and beta receptors was conducted using 1:25 dilution of anti-PDGF alpha and beta receptors monoclonal antibodies respectively followed by incubation in 100 ml of protein A/G Agarose beads for 2 hours at 4^oC. The protein was then washed with TSA buffer and denatured by boiling for 10 minutes in 2X SDS. Equal amounts of immunoprecipitates were run on a 10% acrylamide SDS gels and transferred onto a Hybond nitrocellulose membranes (Amersham, Life Sciences Inc., Arlington Heights, IL). The membranes were blocked with 5% milk in 0.05% Tween in TBS (T-TBS) washed, and incubated with 1:1000 dilution of anti-PDGF alpha and beta receptor antibodies overnight. The membranes were then incubated with 1:2000 dilution of horseradish peroxidase-conjugated secondary antibody in 1% milk in T-TBS for 2 hours. Protein bands were visualized using Amersham’s enhanced chemiluminesence substrate (ECL) and exposure to X-ray film.

[Frontiers in Bioscience 3, a16-22, February 15, 1998]
Reprints
PubMed
CAVEAT LECTOR

EFFECT OF TGF-BETA 1 ON PDGF RECEPTORS EXPRESSION IN HUMAN SCAR FIBROBLASTS

Messadi DV^1,2 , Le A¹, Berg S¹, Huang G¹, Zhuang W¹ and Bertolami CN³

¹School of Dentistry, ²Dental Research Institute, University of California, Los Angeles, California, ³School of Dentistry,University of California, San Francisco, California, USA

Received 1/8/98 Accepted 2/3/98

3. MATERIALS AND METHODS

The effect of TGF- beta 1 treatment on PDGF alpha and beta receptor cell surface expression for NSk, NSc and keloid fibroblasts was analyzed using RadioImmunoBinding assay. All cells were serum starved for 24 hours; one half of the cultures were then preincubated for 24 hours with 10 ng/mL of TGF- beta 1. Using monoclonal antibodies specific for PDGF alpha and beta receptors, the TGF- beta 1 stimulatory effect on the different cell lines was examined.

3.1 Cell cultures

Three replicate cultures each of human NSk, NSc and keloid fibroblasts were cultured as previously described (14). Patients ages ranged from 18 to 50 years. Normal skin tissue samples were matched for age, race and sex. In brief, freshly biopsied tissues, were obtained from the operating room of the University of California Medical Center, Los Angeles (UCLA) and Martin Luther King Hospital, Los Angeles as part of therapeutic procedures. Tissues were enzyme digested, the cells being propagated and maintained in Dulbecco’s Modified Eagle’s Medium (D-MEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin streptomycin. The cells were studied at passages 4-8.

3.2 Addition of exogenous TGF- beta 1

Twenty-four hours before treating cultures with TGF-beta1, regular medium was replaced with serum-free medium. Half of the preconfluent cultures received TGF-beta1 (Sigma Company, St. Louis, MO) at a concentration of 10 ng/mL in serum-free medium for 24 hours. The TGF-beta1-containing medium was replaced with serum-free medium for another 24 hours before harvesting. This dosage and exposure regimen is based on our previous work (13) and the work of Westergren-Thorsson and coworkers (15) showing that TGF-beta1-induced effects on human fibroblast proliferation and activation lasts up to 48 hours after initial exposure.

3.3 Radioimmunobinding assay

Quantitative analysis was performed to determine whether statistically significant differences exist in the expression of PDGF alpha and beta receptors in response to the addition of exogenous TGF-beta1 for the different cell lines. A Dot Microfold apparatus from V&P Scientific, Inc. (San Diego, CA) was used. The different cell lines were fixed with 2% glutaraldehyde and seeded at 5 x 104 cells in the manifold. The manifold has the configuration of a 96 well plate whose bottom surface consists of glass microfilter paper (Whatman, Clifton, NJ). Each well was incubated with 100 ml of the various primary antibodies—either anti-PDGF alpha receptor antibody, PDGF-beta receptor antibody (R&D System Inc., Minneapolis, MN) or mouse IgG as a negative control. The wells were washed several times with PBS+ 2% FBS. Radiolabeled secondary antibody [125I]-conjugated mouse antibody was used at 0.1 mCi/well (ICN, Irvine CA), vacuum applied, and vigorous washing performed to eliminate any nonspecific binding. Wells were cut separately and each was counted for its immune reactivity using a liquid scintillation counter (14). Results were expressed as disintegration per minute (dpm) for each cellular condition and each antibody tested.

3.4 RNA extraction

To determine mRNA levels for PDGF-alpha and beta receptors, fibroblasts from the various tissues were cultured in 75-cm² tissue culture flasks in the presence or absence of TGF-beta1 as described above. At the end of the incubation period, total cellular RNA was extracted from cell cultures using an acid guanidium thiocyanate-phenol-chloroform described by Chomczymski and Saachi (16). Briefly, cells were washed twice with PBS, lysed by adding 10 mL of 4M guanidium thiocyanate buffer, and collected in 50 ml conical tubes. The following solutions were added in succession with vortexing 2-5 minutes each time: 2M sodium acetate pH 4, followed by water saturated phenol, followed by chloroform-isoamyl alcohol. The mixture was centrifuged at 4oC, RNA was ethanol precipitated, centrifuged and resuspended in lysing buffer, centrifuged and the RNA pellet resuspended in 75% ethanol at room temperature. The RNA mixture was then centrifuged, vacuum dried, and the RNA pellet dissolved in 0.5% SDS. Absorbance was checked at OD 260.

3.5 Polymerase chain reaction amplification

Total RNA was isolated from AG 1518 human foreskin fibroblasts (Human Genetic Mutant Cell Repository, Camden, New Jersey) by the method of Chirgwin and coworkers (17). This was used for first strand synthesis in accordance with the work of Claesson-Welch and coworkers (18) who have shown abundant amplification product for PDGF-alpha receptor by this method. RT-PCR was performed using the GeneAmp RNA PCR kit (Perkin Elmer Cetus, Norwalk, CT) according to the manufacturer’s instructions. For cDNA amplification, PCR was performed for 30 cycles. Throughout successive stages of re-amplification and purification, 1.6% Agarose Gels (1XTBE, ethidium bromide) was used to monitor the integrity of the product. The presence of a PDGF-alpha receptor transcript was indicated by the PCR amplification of appropriately sized product generated by the deoxyoligonucleotide primers derived from the published PDGF-alpha cDNA sequences (18) shown to yield a PCR product of 989 bp. PDGF-beta receptor and beta-Actin cDNA probes, the latter used as positive control, were purchased from American Type Culture Collection (Rockville, MD).

3.6 Northern blot analysis

Ten mg of total RNA was separated by electrophoresis in a 0.66 M formaldehyde/1 % agarose gel according to the method of Sambrook and coworkers (19) using a 0.24 to 9.5 kb RNA ladder (BRL, Life Technologies, Inc., Gaithersburg, MD) as size markers. Gels were stained for 10 minutes in ethidium bromide and destained in water. Bands were visible under UV light and then photographed. The agarose gels were transferred to Zeta probe blotting membranes (Bio-Rad Laboratories, Richmond, CA). The membranes were cross- linked by ultraviolet irradiation using a Stratalinker (Stratagene, La Jolla, CA), then prehybridized in the prehybridization buffer containing 50% deionized formamide, 5X Denhardt’s solution, 1% sodium dodecyl sulfate (SDS), 5X SSPE and 100 mg/ml herring sperm DNA. Membranes were prehybridized for 6 hours at 42oC, then hybridized overnight at 42oC with the different probes which were labeled using a random primed labeling kit (Boehringer Mannheim Biochemicals, Indianapolis, IN) according to the manufacturer’s protocol with [alpha-32P] dCTP (-3000 Ci/mmol, Amersham Corporation, Arlington Heights, IL.) in the same prehybridization buffer but without the herring sperm DNA. Membranes were washed twice in 2X SSPE for 15 minutes at room temperature, twice in 2 X SSPE/2% SDS for 45 minutes at 55oC and twice in 0.1 x SSPE for 15 minutes at room temperature. Then membranes were exposed to Kodak X Omat AR film at –70oC overnight.

3.7 Western blot analysis

Confluent fibroblast cultures derived from NSk, NSc and keloid in the presence or absence of TGF-beta1 as described above, were solubilized in cold lysis buffer containing 10mM Tris-HCl, 150 mM NaCl, (TSA) 5 mM EDTA, 1.2% Triton X-100, 2 mM phenyl methylsulfonyl fluoride (PMSF), 2 mM iodoacetamide, 0.15 U/ml aprotinin, and 10 ug/ml leupeptin. Cell suspensions were sheared using a syringe and centrifuged at 2000 rpm for 5 minutes at 4^oC. Lysate pellets were stored at –70^o C. The protein concentration of the different cell lysates were measured using the BCA protein assay kit (Pierce, Rockford, IL) and analyzed by a spectrophotometer.

Immunoprecipitation of PDGF alpha and beta receptors was conducted using 1:25 dilution of anti-PDGF alpha and beta receptors monoclonal antibodies respectively followed by incubation in 100 ml of protein A/G Agarose beads for 2 hours at 4^oC. The protein was then washed with TSA buffer and denatured by boiling for 10 minutes in 2X SDS. Equal amounts of immunoprecipitates were run on a 10% acrylamide SDS gels and transferred onto a Hybond nitrocellulose membranes (Amersham, Life Sciences Inc., Arlington Heights, IL). The membranes were blocked with 5% milk in 0.05% Tween in TBS (T-TBS) washed, and incubated with 1:1000 dilution of anti-PDGF alpha and beta receptor antibodies overnight. The membranes were then incubated with 1:2000 dilution of horseradish peroxidase-conjugated secondary antibody in 1% milk in T-TBS for 2 hours. Protein bands were visualized using Amersham’s enhanced chemiluminesence substrate (ECL) and exposure to X-ray film.