ARTICLE |
Correspondence to: Ulpu K. SaarialhoKere, Dept. of Dermatology, Helsinki U. Central Hospital, Meilahdentie 2, 00250 Helsinki, Finland.
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Summary |
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We studied the expression and regulation of TIMP-3, a recently cloned member of the tissue inhibitor of the metalloproteinase family, during human fetal development and in various human tissues, with emphasis on epithelial structures. Expression of TIMP-3 mRNA was detected by in situ hybridization in developing bone, kidney, and various mesenchymal structures. At 16 weeks of gestation, ectoderm-derived cells of hair germs expressed TIMP-3 mRNA, and beginning from the twentieth week consistent expression was detected in epithelial outer root sheath cells of growing hair follicles. In normal adult human skin, expression of TIMP-3 mRNA was limited to hair follicles, starting at the early anagen (growing) phase and vanishing at the catagen (regressing) phase. TIMP-3 mRNA was not detected in benign hair follicle-derived tumors but was present in tumor cells of infiltrative basal cell carcinomas and in surrounding stromal cells in squamous cell carcinomas. Human primary keratinocytes in culture expressed TIMP-3 mRNAs, the levels of which were upregulated by transforming growth factor-ß (TGF-ß), whereas interleukin-1ß (IL-1ß) and tumor necrosis factor- (TNF-
) had no effect. Our results suggest a role for TIMP-3 in connective tissue remodeling during fetal development, hair growth cycle, and cancer progression. (J Histochem Cytochem 46:437447, 1998)
Key Words: carcinogenesis, extracellular matrix, hair, TGF-ß
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Introduction |
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THE MATRIX metalloproteinases (MMPs, matrixins) are a family of structurally related neutral proteinases involved in the remodeling of extracellular matrix (ECM) in processes such as fetal development, wound healing, inflammation, and tumor invasion (
TIMP-3 mRNA was first detected in human breast tumors (
TIMP-3 is induced in response to mitogenic stimulation and is regulated during normal cell cycle progression (
To explore the physiological role of TIMP-3 in human tissues, we have determined its spatial and temporal expression during fetal development. We have also examined its expression in adult skin and various organs of the body, with special emphasis on epithelial components based on previous data on mouse tissues (. Our results substantiate the role of TIMP-3 in development, hair growth cycle, and tumor growth.
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Materials and Methods |
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Tissues
Formalin-fixed, paraffin-embedded specimens were obtained from the Departments of Dermatology and Pathology, University of Helsinki, Finland. All fetal material originated from medical abortions and was obtained from the Department of Pathology, University of Oulu, Finland. Fetal age was estimated by menstrual age and histological examination. This study was approved by the ethics committee of the Department of Dermatology, Helsinki, Finland. The following subgroups of histological sections were examined.
Probes
A 518-BP fragment corresponding to positions 382900 from the 5' end of the human TIMP-3 cDNA (
In Situ Hybridization
In vitro transcribed anti-sense and sense RNA probes were labeled with [35S]-UTP. Sections were hybridized with probes (2.54 x 104 cpm/µl of hybridization buffer) and were washed under stringent conditions, including treatment with RNase A, as described (
Keratinocyte Cultures
Primary cultures of normal human epidermal keratinocytes were established from skin specimens from a woman undergoing mammoplasty for nonmalignant disease, as described previously ( (20 ng/ml) (both from Boehringer Mannheim; Mannheim, Germany) and bovine TGF-ß2 (5 ng/ml) (kindly provided by Dr. David R. Olsen, Celtrix Co., Santa Clara, CA).
HaCaT cells, transformed human epidermal keratinocytes (obtained from Dr. Norbert Fusenig, DKFZ, Heidelberg, Germany) (
RNA Analysis
Total cellular RNA was isolated from primary keratinocyte cultures using the guanidine thiocyanatecesium chloride method (-32P]-dCTP using random priming and [32P]-cDNAmRNA hybrids were visualized by autoradiography. The mRNA levels were quantitated by densitometric scanning of the X-ray films on gray scale with background subtraction using MCID software (Imaging Research; St Catharines, Ontario, Canada), and corrected for the levels of rRNA visualized by ethidium bromide staining.
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Results |
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TIMP-3 mRNA Is Expressed in Fetal Kidney, Cartilage, Bone, and Mesenchyme
The youngest fetal sample studied was an embryo 7 weeks of age whose main organ systems had developed. Epidermis, seen as a single layer of cells, showed no signal for TIMP-3 mRNA, but expression was detected in neuroepithelium (data not shown), in developing kidney, and in the adjacent gonadal ridge (Figure 1A, Figure 1B, and Figure 1D), and in cells within mesenchymal tissues (data not shown). Developing bone, heart, liver, and lungs, as well as bronchial epithelium, remained negative. At 89 weeks of gestation, chondrocytes of hand and foot plate cartilages (Figure 2A and Figure 2B) and vertebral bodies (data not shown) expressed TIMP-3 mRNA. In other organs the distribution of TIMP-3 mRNA remained the same. At 10 weeks, signal was again detected in chondrocytes of ribs and limbs, and surrounding mesenchymal cells were positive (data not shown). At 12 weeks of gestation, expression of TIMP-3 mRNA was seen in hypertrophic chondrocytes of developing ribs and in surrounding mesenchymal cells that become osteoblasts (Figure 2C and Figure 2D). In the same sample, bones that were already undergoing ossification had TIMP-3-expressing osteoblasts within the newly formed bone matrix (data not shown). Unlike in mouse embryos (
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At 7 and 89 weeks of gestation, no expression of TIMP-1 mRNA was detected. However, at 12 weeks mesenchymal cells surrounding hypertrophic cartilage (Figure 2E and Figure 2F) were positive. In addition, osteoblasts within the bone matrix showed intense signal, as described earlier (
Outer Root Sheath Cells of Hair Follicles Express TIMP-3 mRNA at the Anagen Phase
Biopsies of armpit and scalp skin at 16 weeks of gestation showed hair germs and early hair buds penetrating the underlying dermis. Epidermis was devoid of signal, but epithelial cells of the hair germs showed expression of TIMP-3 mRNA (Figure 3A, Inset a). By 20 weeks, follicles of the scalp showed established morphology, with epithelial cells forming the outer root sheath and mesenchymal cells forming the dermal papilla. Cells of the outer root sheath were consistently positive for TIMP-3 mRNA (Figure 3B, Inset b). In trunk skin, signal was again detected in hair germs and buds, and some follicles already in a more advanced stage of development showed signal in the outer root sheath cells (data not shown). In skin biopsies at 21 and 23 weeks of gestation, outer root sheath cells of each follicle were positive for TIMP-3 mRNA in the biopsies of both scalp and trunk (data not shown).
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In adult hair follicles, TIMP-3 gene was activated during the early anagen (growing) phase and the expression persisted until the follicle entered the catagen phase and started to regress. TIMP-3 mRNA localized mainly to basal cell layer of the outer root sheath in the lower portion of the follicle (Figure 3C, Inset c). In some anagen follicles, a few pale-staining medulla cells within the hair matrix were also positive (Figure 3C). No expression was seen in telogen (resting) follicles (data not shown).
TIMP-3 was not upregulated during re-epithelialization of blistering skin diseases, and expression was not detected in normal epidermis or in sebaceous and sweat glands. However, expression was often seen in fibroblast-like stromal cells surrounding sweat glands and some blood vessels (data not shown). TIMP-1 mRNA was commonly detected in sebaceous glands and in perivascular cells (data not shown) but only occasionally in some outer root sheath cells of anagen hair follicles (Figure 3D). Expression was not detected in fetal hair follicles.
TIMP-3 Expression Is Induced by TGF-ß in Primary Human Keratinocytes
Because TIMP-3 was expressed in hair follicles, we studied its expression in keratinocytes treated with various growth factors known to influence hair growth cycle. Primary human epidermal keratinocytes were treated with TGF-ß2, IL-1ß, and TNF-, and levels of TIMP-3 mRNAs were assayed by Northern blot hybridizations. As shown in Figure 4, epidermal keratinocytes expressed clearly detectable levels of three distinct TIMP-3 mRNAs (2.4, 2.8, and 4.8 KB), and the TIMP-1 probe detected a single 0.9-KB mRNA. Interestingly, treatment of epidermal keratinocytes with TGF-ß2 (5 ng/ml) markedly (4.3-fold) enhanced TIMP-3 mRNA abundance in these cells. In contrast, IL-1ß (5 U/ml) and TNF-
(20 ng/ml) had no marked effect (Figure 4). It has previously been shown that epidermal keratinocytes in culture express TIMP-1 (
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TIMP-3 Is Expressed in Cancer Tissues
To determine whether TIMP-3 also plays a role in the behavior of benign and malignant skin tumors, samples of hair follicle-derived tumors, trichofolliculomas and trichoepitheliomas, and epidermal skin cancers were examined. Interestingly, no signal for TIMP-3 mRNA was detected in the benign tumors (data not shown). Basal cell carcinomas with keratotic (hair-like) differentiation were also negative, whereas in four of five infiltrative basal cell carcinomas TIMP-3 mRNA was detected in tumor cells at the margins of this aggressively growing tumor. In these samples, TIMP-3 mRNA was also detected in areas showing nodular tumor growth (Figure 5A, Inset a) but not in stromal cells. In squamous cell carcinomas, all of which were well- or rather well-differentiated, some stromal cells located diffusively adjacent to the tumor expressed TIMP-3 mRNA (Figure 5C and Figure 5D, Inset c), while malignant cells were negative. There was no correlation between the number of cells expressing TIMP-3 mRNA and the histopathology of the tumor. As in the samples representing normal skin, TIMP-3 mRNA was present in fibroblast-like cells surrounding sweat glands deeper in the dermis.
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Normal gastrointestinal mucosa and mammary gland showed no signal for TIMP-3 mRNA. However, in samples of colon carcinoma intense expression was detected in both macrophages and spindle-like fibroblasts adjacent to the tumor tissue (data not shown). In agreement with the results of
Constitutive expression of TIMP-3 mRNA was also studied in some other organs with epithelial structures. In normal adult kidney, tubule epithelial and glomerular cells expressed TIMP-3 mRNA (Figure 1E and Figure 1F). However, the glandular epithelia of liver, pancreas, parotid gland, testis, and prostate were negative. In addition, bronchial epithelium and adult bronchial cartilage were devoid of signal. The abundance of mRNA in Northern blot hybridizations of these organs (
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Discussion |
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The controlled physiological degradation of the ECM requires a fine balance between MMPs and their inhibitors, whereas in processes such as arthritis and tumor invasion MMP activity predominates, leading to excessive tissue degradation. There are accumulating data on the role of TIMPs and synthetic MMP inhibitors in suppressing tumor growth and invasion. Additional functions independent of metalloproteinase inhibitory activity have also been reported: TIMP-1 and -2 are anti-angiogenic (
Various metalloproteinases and their inhibitors are involved in mammalian development. During the murine peri-implantation period, interstitial collagenase and stromelysin-1 mRNAs are produced by the embryo (
Expression of TIMP-3 was detected throughout fetal hair development, starting from the hair germ stage. Hair germ formation begins in the scalp and face during the third month of gestation and gradually extends in a cephalocaudal direction. The germ consists of a group of epidermal basal cells that protrude into the dermis, forming a hair bud. Mesenchymal cells beneath each bud give rise to the dermal papilla. Further differentiation leads to the formation of the hair cuticle with the surrounding layers of inner and outer root sheath. From the beginning of the fifth month of gestation, different developmental stages are found, ranging from mature follicles to new developing ones ( (TGF-
), and members of the TGF-ß family (
(
had no effect. None of these cytokines markedly affected TIMP-1 expression. TGF-ß2 mRNA co-localizes with TIMP-3 in the basal cells of the outer root sheath (
Immunoreactivity for interstitial collagenase and matrilysin (MMP-7) has been detected in human hair follicles (
In cutaneous squamous cell carcinomas as well as mammary and intestinal carcinomas, TIMP-3 mRNA was detected in stromal cells adjacent to the malignant tumor, which is a common expression pattern for both MMPs and TIMPs. TIMP-3 was not expressed in the benign tumors and the nonaggressive keratotic basal cell carcinomas, but the signal was distinct in the infiltrative subtype and was localized to the malignant cells at the margins of tumor islands. In contrast, both TIMP-1 and -2 are detected only in the stromal cells of surrounding basal cell carcinoma (
In this study, TIMP-3 and -1 were differentially expressed during human fetal development. Furthermore, they were differently regulated in keratinocyte cultures by various growth factors. Further studies are needed to determine the level and specific functions of TIMP-3 protein in various human tissues. On the basis of the expression pattern of TIMP-3 mRNA, TIMP-3 appears to protect the matrix from proteolytic activity and thus regulate normal tissue turnover and inhibit malignant growth. Our results demonstrate a role for TIMP-3 in human fetal development and in remodeling processes of the extracellular matrix.
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Acknowledgments |
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Supported by the Sigrid Jusélius Foundation, the Academy of Finland, the Finnish Cancer Foundation, the Paulo Foundation, the Finska Läkaresällskapet, and grants from Helsinki and Turku University Central Hospitals.
We thank Dr David Carmichael for the TIMP-1 cDNA, Dr Jorma KeskiOja for the HaCaT cells and TGF-ß1, Dr Juha Peltonen for primary keratinocytes, Dr Riitta Herva for pathology expertise, and Ms Alli Tallqvist for excellent technical assistance.
Received for publication December 26, 1996; accepted May 15, 1997.
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