ARTICLE |
Correspondence to: José L. Pablos, Servicio de Reumatología, Hospital 12 de Octubre, 28041 Madrid, Spain.
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tight-skin (Tsk) is a dominant gene mutation that causes a fibrotic skin disease in mice, similar to human scleroderma. Both conditions are characterized by increased numbers of dermal fibroblasts containing high levels of procollagen mRNA. Whether this fibroblast population arises from fibroblast growth or fibroblast transcriptional activation is debated. Proliferation and apoptosis of fibroblasts of normal and Tsk mice were studied in skin sections before, at onset, and in established fibrosis. Tissue sections were immu-nostained with proliferating cell nuclear antigen (PCNA) as proliferation marker. Apoptosis was investigated by in situ end-labeling of fragmented DNA and nuclear staining with propidium iodide. The expression of the apoptosis inhibitor Bcl-2 was investigated by immunohistochemistry. We demonstrate differences in fibroblast proliferation and apoptosis related to postnatal skin growth and development. Neonatal skin exhibits the highest levels of proliferation and apoptosis in fibroblasts. In contrast, low proliferation and absence of apoptosis characterizes adult fibroblasts. Skin fibroblasts express Bcl-2 only in newborns, and at other ages Bcl-2 was restricted to epithelial cells. Our results also suggest that neither increased fibroblast proliferation nor defective apoptosis accounts for the fibrotic phenotype of Tsk. Therefore, transcriptional activation of extracellular matrix genes appears more relevant in the pathogenesis of Tsk fibrosis. (J Histochem Cytochem 45:711-719, 1997)
Key Words: fibroblast, skin, proliferation, apoptosis, scleroderma, Bcl-2, PCNA, TUNEL
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Human scleroderma is characterized by an abnormal accumulation of extracellular matrix components, primarily collagen Types I and III, in the connective tissue (
The kinetics of fibroblast growth have been extensively studied in vitro (
The tight skin mouse (Tsk) is the result of a mutation on the fibrillin-1 gene in chromosome 2 (
In this article we describe the proliferative and apoptotic rates of skin fibroblasts through normal mouse postnatal skin development and Tsk skin fibrosis. Proliferation was evaluated by proliferating cell nuclear antigen (PCNA) staining. PCNA is expressed in proliferating fibroblasts at the G1/S-phase transition, and its expression in fibroblasts in vitro parallels their proliferative ability (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals
TSK (C57BL/6-Tsk+/+pa) and normal (C57BL/6-+pa/+pa) mice were originally obtained from the Jackson Laboratory (Bar Harbor, ME). A Tsk colony has been established and maintained at our animal facility by brother-sister mating of the C57BL/6-Tsk+/+pa by normal (C57BL/6-+pa/+pa) mice. Tsk and normal mice at <1 day, 16 days, and 1 year of age were sacrificed under a CO2 atmosphere. Interscapular skin was removed and portions snap-frozen (liquid N2) for DNA extraction, or fixed for 1 hr at 4C in 4% paraformaldehyde in PBS and paraffin-embedded. Several sections of three paired normal and Tsk littermates at each age were used for quantitative analysis.
Histological Methods
Skin samples from Tsk and normal littermates were cut at 6 µm and mounted on silanized slides. Slides were dewaxed in xylene, rehydrated through an ethanol series and finally, after staining, dehydrated and mounted in Permount medium. Nuclear morphology was evaluated by propidium iodide (PI) and conventional hematoxylin staining. PI staining was performed at 1 µg/ml for 15 min at room temperature (RT) after digestion with DNAse-free RNAse A, 200 µg/ml in PBS for 1 hr at 37C.
PCNA and Bcl-2 Immunohistochemistry
Skin sections were treated with 1.2% H2O2 in absolute methanol for 30 min and stained by the indirect avidin-biotin-horseradish peroxidase method (ABC standard; Vector Laboratories, Burlingame, CA). Color development with diaminobenzidine (Vector Laboratories) was monitored by appearance of the normal Bcl-2 and PCNA brown staining in normal epidermis. The primary antibody to PCNA (PC10; Boehringer Mannheim, Mannheim, Germany) was applied at 1:1000 concentration overnight at 4C. This was the optimum of the tested concentrations (1:100-1:2000) in preliminary experiments. Anti-Bcl-2 (N-19; Santa Cruz Biotechnology, Santa Cruz, CA) was a specific rabbit polyclonal and was applied at 1:500 dilution. Negative controls without primary antibody were included.
Sections were counterstained with hematoxylin for quantification of stained and non-stained fibroblasts. PCNA and Bcl-2 indexes were calculated as the number of positive fibroblasts divided by the total number of fibroblasts x 100, and were evaluated by counting at least 1000 fibroblasts by two independent observers (one of them blinded).
In Situ Detection of Apoptotic Cells
We performed the terminal transferase-mediated dUTP end-labeling technique (TUNEL) as described by Gavrieli, with minor modifications (
DNA Extraction and Electrophoresis
Skin was ground under liquid N2 and digested with proteinase K (200 µg/ ml) (Boehringer Mannheim) in lysis buffer (10 mM Tris-HCl, pH 8, 10 mM EDTA, 0.1 M NaCl, 2% sodium dodecyl sulfate, and 39 mM dithiothreitol) at 56C overnight. The lysate was extracted with phenol/chloroform, ethanol-precipitated, and resuspended in 10 mM Tris-HCl, 1 mM EDTA, pH 8. DNA was treated with 50 µg/ml DNAse-free RNAse-A, re-extracted with phenol/chloroform, and ethanol-precipitated. DNA concentration was determined by spectrophotometry at 260 nm. Twenty µg of DNA was electrophoresed on 1.2% agarose gels in 1 x standard TAE buffer with DNA molecular weight standards (Gibco BRL; Gaithersburg, MD). Gels were stained with ethidium bromide and visualized under UV light.
Statistical Methods
Mean and SD from pooled data of different sections from three Tsk or normal animals at each age were calculated. Student's t-test was used to evaluate the statistical significance of differences between Tsk and normal mice. p values above 0.05 were considered not significant. Interobserver variability was found to be not significantly different.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
PCNA Staining
Both normal and Tsk skin at all ages studied revealed PCNA-stained nuclei in many epidermal keratinocytes, only at the basal and first suprabasal layers (Figure 1). This specific pattern was used as internal control for staining specificity in the evaluated sections, because the proliferative pattern of normal mouse skin fibroblasts was unknown. A high proportion of epithelial cell nuclei in hair follicles and glandular appendages was also stained. No differences in this pattern were observed between Tsk and normal mice. The proportion of labeled nuclei of epidermal and hair follicle cells (proliferative index) decreased slightly in older epidermis. Negative controls without primary anti-PCNA antibody did not show any nuclear staining, and only mast cells showed cytoplasmic staining due to nonspecific binding of avidin (Figure 1G).
|
Stained fibroblast nuclei were observed at all ages in both Tsk and normal mice. The spatial distribution was even in all skin layers. No accumulation of PCNA-stained fibroblasts in any particular area of dermis or hypodermis was detected. A high fibroblast proliferative index was found in newborn skin (<1 day old) (Figure 1A and Figure 1B), decreased in the 16-day-old mouse (Figure 1C and Figure 1D), and was very low in the 1-year-old mouse (Figure 1E and Figure 1F). Tsk and normal mice displayed a similar fibroblast proliferative index at all stages (Figure 2). Statistically significant differences were not detected.
|
Apoptosis
Condensed chromatin, forming pyknotic or crescent-shaped nuclei, and nuclear apoptotic bodies were scarce in dermal fibroblasts of newborn skin and absent in older samples by hematoxylin staining (Figure 3A) and PI staining (Figure 3B). These nuclei were mainly located in the dermis as isolated structures or small clusters. Newborn dermal fibroblasts were also labeled by TUNEL (Figure 3D, Figure 4A, and 4B). TUNEL-stained slides were used for quantification. Quantification of TUNEL-stained fibroblasts nuclei as a proportion was difficult because the figures were under 1/1000 cells. Therefore, we obtained the data as the absolute number of stained nuclei per field at a magnification of x400. Comparison of means failed to show statistically significant differences between Tsk and normal skin (Tsk 0.8 ± 0.33, normal 0.7 ± 0.45 labeled nuclei per field; p=0.57). The TUNEL-negative controls, without TdT, did not show nuclear labeling (Figure 3C).
|
|
TUNEL-labeled nuclei appeared at all ages in epithelial cells of epidermis and hair follicles in much larger numbers than in fibroblasts (Figure 4A-D). Interfollicular epidermal keratinocytes were preferentially labeled in the upper layers, and basal cells were not labeled. Labeling of hair follicle epithelial cells was minimal in hairless newborn skin and maximal at 16 days of age after emergence of hair. Nuclei were selectively labeled in the inner root layer of the medium third of hair follicles (Figure 4C and Figure 4D). By DNA extraction and electrophoresis, we detected the DNA ladder diagnostic of apoptosis only in the 16-day-old skin samples (Figure 5). This coincides with a high number of hair follicle TUNEL-stained cells and a high density of hair follicles at this age.
|
Bcl-2 Staining
Granular cytoplasmic Bcl-2 staining with mild perinuclear condensation in basal epidermal and appendageal epithelial cells was observed in Tsk and normal mice skin at all ages (Figure 4E). In newborn skin, cytoplasmic staining of many fibroblasts in the intermediate layers of the dermis was also seen (Figure 4F). The pattern was similar in Tsk and normal mouse skin. No statistically significant differences were detected between Tsk and normal mouse skin in the index of Bcl-2 labeled fibroblasts (normal 31 ± 6, Tsk 28 ± 7; p=0.31). In the 16-day-old (Figure 4E) and older normal or Tsk mice, there was no Bcl-2 staining in dermal fibroblasts or other connective tissue cells.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In vitro, fetal and neonatal skin fibroblasts have greater proliferative capacity than adult or aged samples (
In mouse, maximal proliferation was seen in the newborn and it decreased as early as the third postnatal week, when the dermis has reached its maximal thickness. This sequence closely resembles the rapid decrease in numbers of procollagen gene-expressing cells that we have previously reported within this time frame (-receptor (
In human scleroderma, it is hypothesized that vascular or inflammatory cell mediators may selectively induce proliferation of the high collagen-producing fibroblasts present in normal dermis. We failed to detect differences in fibroblast proliferation between normal and Tsk fibrotic dermis. Nevertheless, Tsk skin fibrosis shows some differences from its human counterpart. First, it lacks the vascular and inflammatory lesions of human scleroderma (
Apoptosis is related to high cell proliferation under many circumstances and has been regarded as a protective mechanism against the proliferation of partially damaged or wrongly programmed cells. Immortalized and primary embryo fibroblast cell lines with constitutive c-myc expression are susceptible to apoptosis in vitro (
One factor involved in the resistance to apoptosis of senescent fibroblasts is Bcl-2 (
Although abnormal fibroblasts growth responses to different stimuli have been reported in human scleroderma (1(I) gene promoter, caused by a decrease in the binding of a transcriptional inhibitor to AP-1 sites, has been described (
![]() |
Acknowledgments |
---|
Supported in part by grants (94/0235 and 96/5254) from the Fondo de Investigaciones Sanitarias, Ministerio de Sanidad y Consumo, Spain.
Received for publication September 9, 1996; accepted December 5, 1996.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bailey AJ, Black CM (1988) The role of connective tissue in the pathogenesis ofscleroderma. In Jayson M, Black CM, eds. Systemic Sclerosis: Scleroderma. New York, John Wiley & Sons, 75-105
Botstein GR, Sherer GK, LeRoy EC (1982) Fibroblast selection in scleroderma: an alternative model of fibrosis. Arthritis Rheum 25:189-195[Medline]
Bruce SA, Deamond SF, Tsó POP (1986) In vitro senescence of Syrian hamster mesenchymal cells of fetal to aged adult origin. Inverse relationship between in vivo donor age and in vitro proliferative capacity. Mech Ageing Dev 34:151-173[Medline]
Chang CD, Phillips P, Lipson KE, Cristofalo VJ, Baserga R (1991) Senescent human fibroblasts have a post-transcriptional block in the expression of the proliferating cell nuclear antigen gene. J Biol Chem 266:8663-8666
Evan GI, Wyllie AH, Gilbert CS, Littlewood TD, Land H, Brooks M, Waters CM, Penn LZ, Hancock DC (1992) Induction of apop-tosis in fibroblasts by c-myc protein. Cell 69:119-128[Medline]
Fleischmajer R, Perlish JS, Krieg T, Timpl R (1981) Variability in collagen and fibronectin synthesis by scleroderma fibroblasts in primary cultures. J Invest Dermatol 76:400-403[Abstract]
Gavrieli Y, Sherman Y, Ben-Sasson SA (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119:493-501[Abstract]
Goldring SR, Stevenson ML, Downie E, Krane SM, Korn JH (1990) Heterogeneity in hormone responses and patterns of collagen synthesis in cloned dermal fibroblasts. J Clin Invest 85:798-803[Medline]
Green MC, Sweet HO, Bunker HE (1976) Tight skin, a new mutation of the mouse causing excessive growth of connective tissue and skeleton. Am J Pathol 82:493-507[Abstract]
Hoang AT, Cohen KH, Barret JF, Bergstrom DA, Dang CV (1994) Participation of cyclin A in Myc-induced apoptosis. Proc Natl Acad Sci USA 91:6875-6879[Abstract]
Hockenbery DM, Zutter M, Hickey W, Nahm M, Korsmeyer SJ (1991) BCL2 protein is topographically restricted in tissues characterized by apoptotic cell death. Proc Natl Acad Sci USA 88:6961-6965[Abstract]
Ignotz RA, Endo T, Massagué J (1987) Regulation of fibronectin and type I collagen mRNA levels by transforming growth factor-ß. J Biol Chem 262:6443-6446
Ishikawa O., LeRoy EC, Trojanowska M (1990) Mitogenic effect of transforming growth factor ß1 on human fibroblasts involves the induction of platelet-derived growth factor receptors. J Cell Physiol 145:181-186[Medline]
Jimenez SA, Williams CH, Myers JC, Bashey RI (1986) Increased collagen biosynthesis and increased expression of type I and type III procollagen genes in tight skin (Tsk) mouse fibroblasts. J Biol Chem 261:657-662
Jimenez SA (1988) Experimental models of scleroderma. In Jayson M, Black CM, eds. Systemic Sclerosis: Scleroderma. New York, John Wiley & Sons, 119-132
Kahari VM, Sandberg M, Kalimo H, Vuorio T, Vuorio E (1988) Identification of fibroblasts responsible for increased collagen production in localized scleroderma by in situ hybridization. J Invest Dermatol 90:664-670[Abstract]
Kahari VM, Vuorio T, Nanto-Salonen K, Vuorio E (1984) Increased type I collagen mRNA levels in cultured scleroderma fibroblasts. Biochim Biophys Acta 781:183-186[Medline]
Korn JH, Torres D, Downie E (1983) Fibroblast prostaglandin E2 synthesis. Persistence of an abnormal phenotype after short-term exposure to mononuclear cell products. J Clin Invest 71:1240-1246[Medline]
Leof EB, Proper JA, Goustin AS, Shipley GD, DiCorleto PE, Moses HC (1986) Induction of c-sis mRNA and platelet-derived growth factor-like activity by transforming growth factor type ß: a proposed model for indirect mitogenesis involving autocrine activity. Proc Natl Acad Sci USA 83:2453-2457[Abstract]
LeRoy EC (1974) Increased collagen synthesis by scleroderma skin fibroblasts in vitro. J Clin Invest 54:880-889[Medline]
LeRoy EC, Mercurio S, Sherer GK (1982) Replication and phenotypic expression of control and scleroderma human fibroblasts: responses to growth factors. Proc Natl Acad Sci USA 79:1286-1290[Abstract]
Martin GM, Sprague CA, Epstein CJ (1970) Replicative life-span of cultivated human cells: effects of donor's age, tissue, and genotype. Lab Invest 23:86-92[Medline]
Needelman BW, Ordonez JV, Taramelli D, Alms W, Gayer K, Choi J (1990) In vitro identification of a subpopulation of fibroblasts that produces high levels of collagen in scleroderma patients. Arthritis Rheum 33:842-852[Medline]
Pablos JL, Everett ET, Harley R, LeRoy EC, Norris JS (1995) Transforming growth factor-ß1 and collagen gene expression during skin development and fibrosis in the Tsk mouse. Lab Invest 72:670-678[Medline]
Philips N, Bashey RI, Jimenez SA (1995) Increased 1(I) procollagen gene expression in tight skin (TSK) mice myocardial fibroblasts is due to a reduced interaction of a negative regulatory sequence with AP-1 transcription factor. J Biol Chem 270:9313-9321
Polakowska RR, Piacentini M, Bartlett R, Goldsmith LA, Haake AR (1994) Apoptosis in human skin development: morphogenesis, periderm, and stem cells. Dev Dyn 199:176-188[Medline]
Scharffetter K, Lankat-Buttgereit B, Krieg T (1988) Localization of collagen mRNA in normal and scleroderma skin by in situ hybridization. Eur J Clin Invest 18:9-17[Medline]
Schneider EL, Mitsui Y (1976) The relationship between in vitro cellular aging and in vivo human age. Proc Natl Acad Sci USA 73:3584-3588[Abstract]
Siracusa LD, McGrath R, Ma Q, Moskow JJ, Manne J, Christner PJ, Buchberg AM, Jimenez SA (1996) A tandem duplication within the fibrillin 1 gene is associated with the mouse tight skin mutation. Genome Res 6:300-313[Abstract]
Stewart CA, Dell'Orcco RT (1992) Age related decline in the expression of proliferating cell nuclear antigen in human diploid fibroblasts. Mech Ageing Dev 66:71-80[Medline]
Trojanowska M, Wu L, LeRoy EC (1988) Elevated expression of c-myc proto-oncogene in scleroderma fibroblasts. Oncogene 3:477-481[Medline]
Varga J, Rosenbloom J, Jimenez SA (1987) Transforming growth factor ß (TGF-ß) causes a persistent increase in steady state amount of type I and type III collagen and fibronectin mRNAs in normal dermal human fibroblasts. Biochem J 247:597-604[Medline]
Wang E (1995) Senescent human fibroblasts resist programmed cell death, and failure to suppress bcl2 is involved. Cancer Res 55:2284-2292[Abstract]
Wu X, Levine AJ (1994) p53 and E2F cooperate to mediate apoptosis. Proc Natl Acad Sci USA 91:3602-3606[Abstract]
Yamakage A, Kikuchi K, Smith EA, LeRoy EC, Trojanowska M (1992) Selective upregulation of platelet-derived growth factor receptors by transforming growth factor ß in scleroderma fibroblasts. J Exp Med 175:1227-1234[Abstract]