TGF-β1 Antisense Impacts the SMAD Signalling System in Fibroblasts from Keloid Scars

Materials and Methods

Immunohistochemistry. Tissue specimens from 9 patients with keloid scars after otoplasty and control biopsies of normal healthy tissue (from the same patient) were obtained during surgery, which was performed at the Department of Otolaryngology, Head and Neck Surgery, University Hospital of Mannheim. The study protocol was approved by the ethics committee of the institution, and written consent was obtained from all subjects for all procedures. A sample from each resected scar was sent to the pathology laboratory for histologic processing and confirmation of the clinical diagnosis. Samples were frozen in liquid nitrogen for SMAD identification. For in vitro analysis dermal fibroblasts isolated from keloids and normal controls were cultured in Falcon petri dishes (Greiner, Germany) at 37°C in a 5% CO₂ fully humidified atmosphere in serum-free Fibroblast Growth Medium (PromoCell, Heidelberg, Germany) supplemented with antibiotics (Gibco BRL Life Technologies Inc., Gainthersburg, MD, USA). The immunohistochemistry for SMAD2, SMAD3, SMAD4, and SMURF2 was performed using the streptavidin-biotin complex procedure (Amersham, Braunschweig, Germany). After blockage of endogenous peroxidase with 0.3% hydrogen peroxidase for 30 min the sections were washed with phosphate-buffered saline (PBS) and incubated with normal rabbit serum in PBS for 30 min at room temperature in order to block non-specific antibody reaction. Sections were then incubated overnight at 4°C with the primary antibody. The slides were washed in several changes of PBS and then incubated with a peroxidase-conjugated secondary antibody (DAKO, Hamburg, Germany). After being washed twice in PBS, the sections were treated with a streptavidin-biotin-peroxidase complex and peroxidase reaction was performed using Diaminobenzidine DAB (DAKO) as chromogen. Different antibodies were diluted to the desired concentrations in PBS. Controls were carried out by omitting the primary antibody. Light microscopical investigation was performed using a Zeiss Axiophot microscope (Zeiss, Oberkochen, Germany).

Oligodeoxynucleotides. Phosphorithiotated 14-mer oligodeoxynucleotides (ODN) were synthesised on an Applied Biosystems 394 DNA synthesiser (Applied Biosystems Inc., Foster City, CA, USA) by means of B-cyanothylphosphoramidite chemistry to minimise degradation by endogenous nucleases. The antisense oligonucleotide (5′-CGA TAG TCT TGC AG-3′) was directed against the translation start site and surrounding nucleotides of the human TGF-β1 cDNA. The in vitro inhibitory effect of these antisense ODNs on TGF-β1 expression at both the mRNA and protein level in human cells had been described previously (19). All experiments were performed with 3 μM ODNs. To determine the effect of oligonucleotides on the expression of SMAD mRNAS or SMURF2 mRNA, fibroblasts were plated at a density of 105 cells/microtiter well in 24 well polystrene plates (Falcon; Becton Dickinson Labware, Franklin Lakes, NJ, USA). After 24 hours, the cells were rinsed twice with medium and then fresh TGF-β1 oligo medium containing antisense ODNs was added, followed by an incubation period of 48 h.

RT-PCR. To isolate the RNA from the fibroblasts grown in monolayer, the cells were directly lysed in the culture dish by addition of 1 ml RNA-Clean (RNA-Clean System, AGS, Heidelberg, Germany). After addition of 0.2 ml chloroform per 2 ml of homogenate and centrifugation for 15 minutes at 12,000 ×g (4°C), the supernatant was removed from the RNA precipitate. The RNA pellet was washed twice with 70% ethanol by vortexing and subsequent centrifugation for 8 minutes at 7,500 ×g (4°C). After drying the RNA pellet, it was dissolved in DEPC water. The RNA was reverse transcribed (StrataScript First-Strand Synthesis System, Stratagene, La Jolla, CA, USA) into cDNA using random-oligunucleotide primers. Primer sequences used for RT-PCR were as follows: for SMURF2: sense 5′-GTT GTG ATG GGT TCT GAT TC-3′ and antisense 5′-CAC CAA TGG CAA AAG GCT-3′; for SMAD2: sense 5′-GGA GCA GAA TAC CGA AGG CA-3′ and antisense 5′-CTT GAG CAA CGC ACT GAA GG-3′; for SMAD3: sense 5′-AGA AGA CGG GGC AGC TGG AC-3′ and antisense 5′-GAC ATC GGA TTC GGG GAT AG-3′; for SMAD4: sense 5′-CAT CGA CAG AGA CAT ACA G-3′ and antisense 5′-CAA CAG TAA CAA TAG GGC AG-3′ and for SMAD6: sense 5′ CAA GCC ACT GGA TCT GTC CGA-3′ and antisense 5′-TTG CTG AGC AGG ATG CCG AAG-3′. SMAD and SMURF2 mRNA levels were measured in all cell types using RT-PCR (MMP-CytoXpress Multiplex PCR Kit, Bio Source, San Francisco, CA, USA) according to the manufacturer's instruction manual. To fractionate the MPCR DNA products, the MPCR products were mixed with 6× loading buffer and separated on a 2% agarose gel containing 0.5 mg/ml ethidium bromide, visualised with UV light and recorded using a CCD camera. To test the quality of the cDNA, the kit included primers for GAPDH. Results were obtained in two independent experiments.