1 Lankenau Institute for Medical Research, 100 Lancaster Avenue, Wynnewood, PA 19096, USA
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Abstract |
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Abbreviations: DMBA, 7,12-dimethylbenz[a]anthracene; KC, keratinocyte colony; TPA, 12-O-tetradecanoylphorbol-13-acetate.
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Introduction |
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Several lines of evidence suggest that clonogenic keratinocytes (KCs) are closely related, or identical to multipotential stem cells. Most KCs originate from the bulge-region of the hair follicle (1012). The bulge-region has low mitotic and DNA synthetic activity in vivo (10). In contrast, the highly mitotic matrix region of the hair follicle has extremely low clonogenic activity in vitro (10). Therefore, most mitotically active keratinocytes in vivo probably are not clonogenic in vitro. It is also suggested that keratinocyte colonies take their origin from quiescent cells. The three-dimensional reconstruction of hair follicle from serial sections made 14 months after the complete labeling of the epidermis in neonatal mice disclosed the presence of highly persistent label-retaining cells associated with the bulge region in hair follicles (13). Slow cycling (label-retaining) epidermal cells in vivo behave like clonogenic stem cells in vitro (14). Therefore, we conclude that most or all KCs take their origin from stem cells.
We have identified several factors that influence the number of KCs. Although keratinocyte colony (KC) number remains virtually constant throughout most of the adult life of mice (7), the KC number increases during the tumor promotion of initiated skin (7). A second factor influencing KC number is the strain of mice (15). Therefore, we conclude that the amplification of keratinocyte stem cells might play a role in skin carcinogenesis and that KC number can potentially be genetically regulated. We suggest that the identification of regulatory genes involved in stem cell amplification would add to our understanding of skin carcinogenesis.
Recently, we have demonstrated that the mouse KC number is a genetically defined quantitative complex trait (15). We found that strains of mice with different genetic origins have significant differences in the number of KC forming cells (15). In particular, we have demonstrated that the number of KC forming cells is significantly higher in female C57BL/6 mice than in female BALB/c mice. The F1 hybrid of these two parental strains had an intermediate KC number. The KC number was significantly different in the two backcrosses (C57BL/xCB6F1; BALB/cxCB6F1) mice. This observation led us to conclude that the alleles associated with high and low KC numbers were segregated. We have analyzed several models considered for the genetic determination of quantitative traits and have demonstrated that our trait is regulated by two (or more) loci with additive but not necessary equal effect. Finally, we have established that the KC number is genetically defined and a quantitative complex trait.
Therefore, we initiated the present study to identify loci regulating KC number. It follows from our data that mouse skin probably has at least two major subpopulations of KCs that are regulated by different genes. We conducted linkage analysis of KC number in genetic crosses of C57BL/6 (high colony number) and BALB/c (low colony number) for the identification of loci linked with the two populations of keratinocyte stem cells. Our findings were novel and completely unexpected: the loci (one major and several minor), linked to the subpopulation of keratinocytes forming small colonies, were related, if not identical to previously identified loci associated with susceptibility or resistance to skin tumor promotion and tumor development.
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Materials and methods |
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All mice at 7 weeks of age were in the resting stage of the growth cycle of hair follicle as evidenced by their pink skin. All experiments were carried out with the approval of the Lankenau Institutional Animal Care Use and Committee.
Phenotyping
Epidermal cells including those from hair follicles were harvested from the dorsal skin by a trypsinization procedure optimized for reproducible yields and single cell suspension by our laboratory (16) and grown from single cells on irradiated 3T3 (Swiss mouse) fibroblasts. Swiss 3T3 fibroblasts obtained at the 119th passage from the American Type Culture Collection (Rockville, MD, USA) were irradiated with 50 Gy (5000 rads) using a cesium source and then used as feeder cells. Colonies of epidermal cells were cultivated in a supplemented Williams medium (17). Briefly, as supplements we used: 5.0 mg/l of insulin; 10 mg/l transferrin; epidermal growth factor 10 mg/l; 876 mg/l glutamine; 2 mg/l relenyl acetate; 0.2 mg/l ergocalciferol; linoleic acid-BSA; 2 mg/l hydrocortisone; 10 ml/l penicillin/streptomycin; 100 ml/l fetal bovine serum (Hyclone, Defined Logan City, UT); Collagen-fibronectin coating consisting of 100 ml Williams Medium E, 1.0 mg of fibronectin, 10.0 mg bovine serum albumin, 3.0 mg Vitrogen collagen (Cohesion, Palo Alto, CA), and 1.0 ml of i.m. HEPES. The medium was optimized for CD-1 epidermal cells. The cultures were maintained in a humidified incubator at 32°C with 5% CO2. The medium was changed three times weekly beginning on the second day after seeding. After 2 weeks of cultivation, dishes were rinsed twice with Dulbecco phosphate-buffered saline and were fixed in 10% formalin for at least 24 h.
Keratinocytes harvested from each animal in each of our groups were put into four 60 mm plastic dishes (1000 viable cells per/dish). All dishes were fixed with neutral buffered formalin and stained with Rhodamine B. The KC number and size in mm2 were analyzed with Fluor-STM MultiImager (BIO-RAD). The KC number in each dish was counted after 2 weeks of cultivation (15) with colony sizes ranging from 0.1 to 12 mm2. An average KC number in two duplicate dishes from each animal ranging in size from 0.1 to 2 mm2 was designated as the number of small colonies, and from 2.1 mm2 or more as the number of large colonies respectively (Figures 1 and 2). Finally, we had two phenotypes. The first of our phenotypes was the number of small colonies from each individual animal of each segregating crosses, the second, the number of large colonies.
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Genotype analysis
DNA was isolated from the mouse liver by a standard proteinase K procedure (18). Genotyping was performed by polymerase chain reaction (PCR) as previously described (19), using microsatellite primers purchased from Research Genetics (Hunstsville, AL). This investigation used 141 microsatellite markers. These markers were chosen to cover all known chromosomal segments of STSs (sequence-tagged sites) originating in autosomes from C57BL/6 and BALB/c mice. The maximum distance between two adjacent markers was no more than 15 centiMorgans (cM). PCR products were analyzed by electrophoresis through 3% agarose gels, stained with ethidium bromide, and visualized under ultraviolet light. Statistical analyses were performed using the program Qlink kindly provided by Dr Norman Drinkwater, of the University of Wisconsin. Qlink was written to simplify linkage analysis for quantitative trait loci (QTL) using nonparametric methods based on those described by Kruglyak and Lander (20). Loci were ordered using the map locations reported in the 2000 Mouse chromosome Committee Reports obtained from the Mouse Genome Database (http://www.informatics.jax.org/ccr/searches/index.cgi?year=2000).
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Results |
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Genetic mapping of loci affecting the number of small and large KCs
To identify loci that could regulate the number of small and large colonies, we used a procedure similar to that described by Angel et al. (19). A total of 104 intercross mice were initially investigated for clonogenic activity of their keratinocytes (15). We used the same protocol to determine the number of small and large colonies in intercross and backcross mice as has been described (15). The 141 microsatellite polymorphic markers used allowed us to generate a complete genome-wide scan with intervals at ~15 cM when available. In regions where linkage to the number of small and large colonies was detected, the density of markers was increased to obtain a more accurate genetic dissection in the area of interest. Overall, genomic coverage reached about 95.3% across the 19 autosomal chromosomes. The KC number of mice homozygous and heterozygous for each locus was compared by using Qlink based on the Wilcoxon rank test. Qlink was written to simplify linkage analysis for quantitative trait loci (QTL) using nonparametric methods based on those described by Kruglyak and Lander (20). Tables II, III and IV show all of the microsatellite markers positively linked to the number of small or large colonies in the intercross or backcross respectively. Significant association (21) of an increased number of small KCs with the inheritance of the C57BL/6 allele was detected for the region between D9Mit262 and D9Mit236 loci with a single point P = 0.00002 (LOD score = 3.88; genome-wide P value = 0.0139) and P = 0.0002 (LOD score = 3.84; the P value for the whole genome = 0.0153) respectively (Table II
).
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Suggestive evidence (21) for linkage or close to suggestive linkage with the number of small colonies was found with the markers on mouse chromosomes 1, 6, 7, 8 (Tables II, III). On chromosome 1, D1Mit178 locus had a LOD score of 1.65 and a single point P value = 0.0058. An analysis of the 12 markers on this chromosome suggests that a gene influencing the number of small colonies may reside in a 35 cM interval from D1Mit178 to D1Mit132. A third wide region showing an association with a high number of small colonies with C57BL/6 alleles was found on chromosome 6 in the interval between D6Mit93 and D6Mit323. A peak suggestive LOD score (21) of 2.25 (single point P = 0.001) was obtained at D6Mit99. A fourth region with suggestive linkage (21) to a high number of small colonies was identified in the interval between D7Mit66 and D7Mit259. A peak LOD score of 2.16 (single point P = 0.0016) was obtained at D7Mit105 (63.5 cM). A fifth region showing evidence of suggestive linkage (21) was found near the centromere of chromosome 8; a peak LOD score of 1.94 (single point P = 0.002) was obtained at locus D8Mit58. The same result has been obtained in (C57BL/6xCB6F1) backcross where the high number of small colonies linked with alleles C57BL/6 from the same region of chromosome 8 between D8Mit58 and D8Mit339 (Table III
).
Suggestive linkage (21) to the high number of large colonies was obtained for the markers on mouse chromosome 4 (Table IV). D4Mit264 and D4Mit181 had a LOD score 3.05 (with single point P = 0.0001). An analysis of the eight markers on chromosome 4 suggests that the gene determining a high number of large colonies may be situated in the interval between D4Mit264 (1.9 cM) and D4Mit181 (2.5 cM) and is associated with BALB/c alleles (Table IV
). Therefore, we conclude that a gene regulating the high number of large colonies resides in the region of locus D4Mit264. We designated this locus keratinocyte stem cell locus 2 (Ksc2).
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Discussion |
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We performed genetic mapping of two backcrosses and intercross of the parental BALB/c and C57BL/6 mice for the identification of loci associated with the total KC number. We found several loci on the proximal region of mouse chromosomes 1, 4, 8, the proximal and central regions of chromosome 6, the distal region of chromosome 7 and the central region of chromosome 9 associated with the whole KC number (unpublished data). All of these regions had a single point significance with P = 0.050.002. There are several reasons why our results did not reach genome-wide significance. First, the number of KCs might not be genetically regulated. However, in our previous publication (15) we established that KC number is a genetically defined complex trait. Second, more intercross mice might be needed for the analysis. However, a number of investigators (2224) demonstrated that 100 intercross mice might be sufficient to reach genome-wide P value. Finally, we suggest that in each individual dish, we had a mixture of several subpopulations of clonogenic keratinocytes. This was the reason why we decided to investigate the size of the colonies.
We have analyzed KC areas measured with the Fluor-STM MultiImager (BIO-RAD) system and have shown that mouse skin has at least two major subpopulations of clonogenic keratinocytes regulated by different genes. Initially, we found that the KC size is genetically regulated (Table I). Analysis of the frequency distribution of colonies of different sizes (Figure 2
) brought us to the conclusion that mouse epidermis has at least two major subpopulations of clonogenic keratinocytes that can produce small (<2 mm2) or large (from 2.1 to 12 mm2) colonies. The numbers of small and large colonies have demonstrated significant differences between parental strains. This finding led us to conclude that genes regulating numbers of both small and large colonies can be mapped. Moreover, the number of small colonies was higher in BALB/c mice, while large colony number was higher in C57BL/6 mice. Therefore, we suggest that different genes regulated the numbers of small and large colonies. Thus, all of our colonies were separated into small and large, according to their size and a genetic mapping of loci associated with small and large colony number was performed.
We found that our loci on chromosomes 1, 4, 6, 7, 8, 9 linked to total KC number were retained in the analysis of the number of small and large KCs. In particular, we found that the loci on chromosomes 1, 6, 8, 9 and 7 were associated with a number of small colonies (Table II), while the locus on chromosome 4 was linked with a number of large colonies. Moreover, the level of significance for most of these loci increased upon the reanalysis of the phenotype. To this end, we concluded that mouse skin has two major subpopulations of clonogenic keratinocytes that are regulated by different genes.
Multilocus analysis of chromosome 9 suggested that the most likely interval surrounding D9Mit262 (41 cM) and D9Mit236 (43 cM) in the mid-portion of the chromosome contained the gene involved in the regulation of the number of keratinocyte stem cells and was responsible for small KCs. A defined LOD score suggested a single locus (Figure 3; Table II
).
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A locus involved in skin tumor promotion, Psl1, previously mapped to a region of chromosome 9 (25), overlapped our Ksc1 locus. In particular, the inheritance of the DBA/2 allele on mouse chromosome 9 was associated with increased susceptibility to TPA promotion (25). C57BL/6 mice represent extreme resistance to skin TPA tumor promotion. Unfortunately, Angel et al. (25) report the combined data of backcross, intercross and recombinant strains concerning Psl1, making it difficult for us to do a more detailed analysis of Psl1 in association with Ksc1. Moreover, Mock et al. reported that a higher than expected linkage score was seen for the marker D9Mit271 (48 cM) in association with skin tumor susceptibility (26). In this work, the inheritance of the BALB/c allele on the central region of mouse chromosome 9 represented resistance, while the inheritance of SENCARA/Pt alleles of this region of chromosome 9 was associated with sensitivity to papilloma development (26). This locus is also closely related to our Ksc1. We, therefore, suggest that the gene(s) on chromosome 9 regulating the number of small KCs may also affect sensitivity or resistance to skin tumor promotion.
Nagase et al. (24) found two loci [Skts1, Skts2] linked to papilloma development on chromosome 7. The Mus spretus allele of Skts2 (64 cM) locus in the distal region of chromosome 7 is associated with female specific papilloma resistance, while the Mus musculus allele in this locus is associated with susceptibility to papilloma development (24). In our intercross mice, a BALB/c allele of D7Mit105 (63.5 cM) locus showed suggestive association with the number of small colonies in almost the same region as the Skts2 locus. Therefore, the gene regulating small KC number might be involved in the regulation of susceptibility or resistance to papilloma development in the Skts2 locus.
A large region on chromosome 6 that Nagase (24) linked with the susceptibility or resistance to benign and malignant skin tumors surprisingly correlated with our region on chromosome 6 (Table II) found to be associated with a high number of small colonies. In addition, our LOD score peak on chromosome 6 (D6Mit99, 36cM) (Table II
) was situated in the region close to the peak P value on chromosome 6 (D6Mit9, 32cM; D6Mit29, 35 cM) in the Nagase mapping (24). Moreover, the gene for transforming the growth factor alpha (TGF-
) situated on chromosome 6 at 35.8 cM from centromere might influence papilloma development (27) as well as the number of keratinocyte colonies (Morris, unpublished data). We suggest that one gene or group of genes linked with the number of small colonies on chromosome 6, may affect the development of benign and malignant skin tumors. Finally, our major Ksc1 locus on chromosome 9 and two minor suggestive loci on chromosomes 6 and 7 are surprisingly close to loci mapped by other investigators and found to be associated with skin tumor promotion (25), or sensitivity or resistance to skin tumor development (2426). We hypothesize that the population of clonogenic keratinocytes able to produce small colonies represents a specific population of skin stem cells that may be responsible for sensitivity or resistance to skin carcinogenesis. It follows from our data that genes associated with C57BL/6 alleles on chromosomes 6 and 9, and BALB/s allele on chromosome 7 are probably responsible for the population of skin stem cells resistant to skin carcinogenesis. Future investigation of other genetic crosses with strains of mice susceptible to skin carcinogenesis will help us to identify the loci responsible for sensitivity to skin carcinogenesis.
The Ksc1 region on mouse chromosome 9 shares linkage homology with human chromosomes 15q22.1 (MAP2K1), 15q21 (TCF12), 15q21-q22 (ADAM10), 6pter-qter (BMP5), 14pter-qter (COX7A3). Figure 3 shows the map location of chromosome 9 markers used in this study relative to the locations of other genes mapped to this region. The region of the Ksc1 locus contains a cluster of genes responsible for a normal mouse development (Adam10, 4; Foxb1a; Bmp5; Hnf6; Tcf12; Om2a, 2b) and may be involved in the regulation of keratinocyte stem cell number due to their opportunity to influence growth and differentiation (2834). It is interesting that Adam10, situated in our region of chromosome 9, plays a role in the Notch signaling pathway that may be involved in keratinocyte growth arrest and entry into differentiation (35) and may be expressed in the mouse hair follicle (36). It has been shown that Delta-Notch signaling might be involved in stimulating human epidermal stem cell differentiation (37), thus suggesting that Adam 10 may be a candidate gene in the Ksc1 locus.
Another interesting group of genes in this region includes mitogen-activated protein kinases 1 (Map2k1) (38) or Anxa2 that represent a major substrate for growth factor receptor protein-tyrosine kinases and protein kinase C (39). Morris et al. established that the number of clonogenic keratinocytes increases in response to TPA treatment (7) possibly resulting in protein kinase signal transduction. Thus, one or more genes (Map2k1, Anaxa2) might be involved in the regulation of the number of clonogenic keratinocytes.
The Ksc2 locus on mouse chromosome 4 shares linkage homology with human chromosomes 8q13 (LYN, GWM), 8q11 (MOS), 8q13-q21 (GEM). The Ksc2 locus on chromosome 4 is represented in Figure 3. Possible candidate genes for Ksc2 include the Lyn gene, expressed in the skin, which is a member of the Src subfamily and encodes protein tyrosine kinase (40). The Mos genes are regulated posttranscriptionally and expressed in growth and oocyte maturation (41) and may be involved in the regulation of stem cell number. The mitogen-induced gene, Gem, encodes a GTP-binding protein that belongs to a new family within the Ras superfamily (42). The regulated expression pattern of Gem suggests a role for this protein in cellular responses to growth stimulation and may be a candidate gene for the Ksc2 locus.
In summary, at least one gene on chromosome 9 (Ksc1) and suggestive evidence (21) for the Ksc2 locus (chromosome 4) were identified as regulatory loci involved in the formation of small and large colonies respectively. Our data suggest that clonogenic keratinocytes producing small colonies might represent a specific population of keratinocyte stem cells responsible for sensitivity or resistance to skin carcinogenesis. The identification and eventual cloning of the Ksc1 and Ksc2 genes may lead us to the identification of genes regulating keratinocyte stem cell amplification. Moreover, as suggested by our data, the mechanisms regulating the intrinsic number of keratinocyte stem cells undoubtedly underlie the cell responses to external manipulation. The ability to manipulate these genes in vivo raises the exciting possibility for a novel treatment for skin disease and cancer. Furthermore, the keratinocyte stem cell control genes are possibly part of a universal regulatory mechanism for maintaining resting numbers of stem cells or their amplification. Finally, one of the future problems of great interest to us is whether the stem cell regulatory genes might be targets in carcinogenesis caused by environmental chemicals or ultraviolet light.
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Notes |
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3 To whom correspondence should be addressed Email nvp2002{at}columbia.edu
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Acknowledgments |
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References |
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