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. Author manuscript; available in PMC: 2007 Oct 15.
Published in final edited form as: J Invest Dermatol. 2005 Sep;125(3):499–509. doi: 10.1111/j.0022-202X.2005.23844.x

Co-Regulation of p16INK4a and Migratory Genes in Culture Conditions that Lead to Premature Senescence in Human Keratinocytes

Benjamin W Darbro *, Galen B Schneider †, Aloysius J Klingelhutz ‡
PMCID: PMC2020850  NIHMSID: NIHMS22112  PMID: 16117791

Abstract

Cellular stasis, also known as telomere-independent senescence, prevents many epithelial cells from becoming immortalized by telomerase alone. As human keratinocytes age in culture, protein levels of the tumor suppressor p16INK4a continue to increase, resulting in growth arrest independent of telomere length. Differences in culture conditions have been shown to modulate both p16INK4a expression and replicative capacity of human keratinocytes; however, the mechanism of p16INK4a induction under these conditions is unknown. Using multiple primary keratinocyte cell strains, we verified a delay in p16INK4a induction and an extended lifespan of human keratinocytes when grown in co-culture with post-mitotic fibroblast feeder cells as compared with keratinocytes grown on tissue culture plastic alone. Evaluation of gene expression levels in the two culture conditions by microarray analysis, and subsequent validation, demonstrated that keratinocytes cultured on plastic alone had significantly increased expression of many genes involved in keratinocyte migration and reduced expression levels of genes involved in keratinocyte differentiation. Higher levels of p16INK4a expression were present in cells that also displayed increased amounts of autophosphorylated focal adhesion kinase and urokinase plaminogen activator receptor (uPAR), both markers of keratinocyte migration. Furthermore, when tyrosine phosphorylation or urokinase-type plasminogen activator (uPA)/uPAR function was inhibited, both keratinocyte migration and p16INK4a expression were reduced. Our results indicate that keratinocytes cultured in the absence of feeder cells exhibit a migratory phenotype and suggest that p16INK4a is selectively induced under these conditions by a mechanism involving tyrosine kinase activity and the urokinase plasminogen activation system.

Keywords: differentiation, FAK, Microarrays, uPAR

After a finite number of cell divisions, normal human somatic cells eventually enter a non-proliferative state termed senescence (Hayflick and Moorhead, 1961). Following introduction of the catalytic subunit of the enzyme telomerase (hTERT), some cell types, including human fibroblasts, retinal pigment epithelial cells, vascular endothelial cells, and mesothelial cells, can bypass senescence and become immortal (Bodnar et al, 1998; Yang et al, 1999; Dickson et al, 2000). Other cell types, including human keratinocytes, mammary epithelial cells, bladder urothelial cells, and prostatic epithelial cells, appear to experience a telomere-independent senescence or growth arrest, termed cellular stasis, that cannot be overcome by maintaining telomere length alone (Foster and Galloway, 1996; Brenner et al, 1998; Kiyono et al, 1998; Jarrard et al, 1999; Puthenveettil et al, 1999; Dickson et al, 2000; Sandhu et al, 2000; Stampfer and Yaswen, 2003). There is evidence that suggests that some of these epithelial cell types can become immortal by expression of hTERT alone if they are grown in co-culture with post-mitotic fibroblast feeder cells as opposed to being grown on tissue culture plastic alone (Ramirez et al, 2001; Herbert et al, 2002). The mechanism to account for these culture condition-modulated differences in cellular lifespan remains undefined.

Over 90% of all human malignancies arise from epithelial cells; thus, it is of great importance to better understand the mechanisms through which these cell types become immortal. Human keratinocytes are a model epithelial cell type known to give rise to cancers such as squamous and basal cell carcinomas. In vitro, human keratinocytes have been immortalized by telomerase activation, either by ectopic expression of hTERT or expression of the E6 viral oncogene of high-risk human papillomavirus (HPV), and inhibition of the tumor suppressor protein retinoblastoma (Rb), by expression of the E7 viral oncogene of high-risk HPV (Demers et al, 1994; Klingelhutz et al, 1996; Kiyono et al, 1998). Expression of the E7 viral oncogene is capable of extending keratinocyte life span in culture; however, E7 by itself does not generally result in immortalization (Kiyono et al, 1998). Keratinocyte cell strains positive for telomerase activity that have lost expression of the cyclin-dependent kinase (CDK) inhibitor p16INK4a, typically by mutation, deletion, or promoter methylation, are also immortalized (Kiyono et al, 1998; Dickson et al, 2000; Farwell et al, 2000). These findings strongly suggest a role for the p16/Rb pathway in providing a barrier to human keratinocyte immortalization.

The role that p16 inactivation plays in keratinocyte immortalization is further supported by its association with cellular stasis. As keratinocytes age in culture, there is a progressive accumulation of p16 protein. In addition, ectopically expressed p16 can induce premature growth arrest in a variety of cell types including malignant glioma and pancreatic cells, oral squamous cell carcinoma, osteogenic sarcoma, and prostatic epithelial cells (Uhrbom et al, 1997; Timmermann et al, 1998; Dai & Enders, 2000; Calbo et al, 2001; Schwarze et al, 2001). Increased levels of p16 disrupt CDK4 and CDK6/Cyclin D complexes, releasing other sequestered CDK inhibitors such as p21 and p27 from these complexes, ultimately increasing levels of the active, hypophosphorylated form of Rb (Kiyono et al, 1998; McConnell et al, 1999). Through its ability to accumulate as keratinocytes age, it has been hypothesized that p16 is the causative agent that induces telomere-independent cellular stasis in human keratinocytes.

To date, it is unknown what upstream signaling events cause the increased expression of p16 that precedes keratinocyte growth arrest during cellular stasis. Increased cellular stress induced by inadequate culture conditions has been hypothesized to induce p16 expression and consequent telomere-independent cellular stasis (Ramirez et al, 2001); however, the mechanism through which this proposed stress translates into upregulation of p16 in keratinocytes is unclear. It has been shown that p16 is co-expressed with the γ2 chain of laminin 5 at the leading edge of in vitro wounded keratinocytes (Natarajan et al, 2003), suggesting that p16 expression is activated in keratinocytes induced to migrate. The issue of how p16 is induced is of extreme significance given the fact that many epithelial cancers bypass p16-mediated tumor suppression.

To characterize the phenotype associated with culture-induced p16 upregulation, we have used DNA oligonucleotide microarrays to assess the global gene expression patterns between human keratinocytes grown on tissue culture plastic alone or in co-culture with post-mitotic, γ-irradiated fibroblast feeder cells. These two culture conditions were selected since they have been shown to differentially modulate both p16 expression and keratinocyte replicative capacity. Analysis of the microarray data indicated that, compared to keratinocytes co-cultured with feeder cells, keratinocytes grown on plastic alone increase the expression of several genes involved in migration and decrease the expression of genes associated with differentiation. Furthermore, we found that markers of keratinocyte migration, such as focal adhesion kinase (FAK) autophosphorylation and urokinase plasminogen activator receptor (uPAR) expression, are preferentially induced in keratinocytes grown on plastic alone and correlate with p16 induction. Lastly, when tyrosine phosphorylation was inhibited using Herbimycin A, a non-specific inhibitor of tyrosine kinases, or urokinase-type plasminogen activator (uPA)/uPAR function was inhibited by Amiloride, a competitive inhibitor of uPA, both migration and p16 expression were reduced in keratinocytes cultured on plastic alone. These findings suggest that the increased stress experienced by keratinocytes grown on plastic alone is the result of a culture-induced migratory response that is absent or reduced when keratinocytes are grown in co-culture with feeder cells.

Results

Culture conditions alter both replicative capacity and p16 expression

Variable results have been reported concerning both p16 expression and replicative capacity of human keratinocytes cultured on tissue culture plastic alone or in co-culture with post-mitotic fibroblast feeder cells (Ramirez et al, 2001; Rheinwald et al, 2002; Baek et al, 2003; Fu et al, 2003; Kang et al, 2003, 2004). Using three primary strains of human foreskin keratinocytes (HFK) derived from different donors, we found that HFK serially subcultured on plastic alone growth arrested at approximately 16.6 ± 3.9 population doublings (PD). In contrast, HFK grown in co-culture with feeder cells reached approximately 28.1 ± 2.3 PD before proliferation ceased. Statistical analysis using paired Student's t test found this difference to be significant (p = 0.030). Comparing the replicative capacities of each HFK strain, we found that co-culture with feeder cells extended HFK replicative capacity by an average 11.4 ± 3.5 PD. Representative growth curves from the three primary HFK strains are shown in Fig 1A and B. Protein levels of p16 were also altered in HFK grown in the two culture conditions. We observed a steady accumulation of p16 protein in HFK cultured on plastic alone that reached a maximal level at growth arrest (Fig 1C). In contrast, HFK grown in the presence of feeder cells exhibited delayed p16 accumulation that ultimately increased in later passages correlating with growth cessation (Fig 1C). At late passage, an increase in p16 expression was seen in all primary HFK strains cultured with feeder cells (data not shown). Thus, the two culture conditions differed in the ability to induce p16 expression and delay keratinocyte senescence.

Figure 1.

Figure 1

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Lifespan extension and delay in p16 induction in keratinocytes co-cultured with feeder cells. (A) Replicative capacities of three primary human foreskin keratinocyte (HFK) cell strains grown on plastic alone (Strain 1: 12.9 population doublings (PD), Strain 2: 20.7 PD, and Strain 3: 16.3 PD). (B) Replicative capacities of three primary HFK cell strains grown in co-culture with post-mitotic fibroblast feeder cells (Strain 1: 25.8 PD, Strain 2: 28.1 PD, and Strain 3: 30.3 PD). (C) Immunoblot of p16 protein levels in Strain 1 HFK cultured on plastic alone (lanes 1–4) and in co-culture with feeder cells (lanes 5–8). Protein levels of actin are included as a loading control. Approximate PD are represented below each lane.

Microarray analysis and validation of differential gene expression in different culture conditions

To determine which genes and signaling pathways are activated or repressed in association with culture-induced p16 expression, we used Affymetrix oligonucleotide microarrays to assay global gene expression patterns in HFK grown under the two different culture conditions. RNA was collected from two independent, mid-passage strains of HFK serially subcultured under both culture conditions. Following hybridization and detection of transcript abundance on Affymetrix HG-U133A GeneChips, we compared gene expression in HFK cultured on plastic with that of HFK in co-culture with feeder cells. The change in gene expression level was considered significant if (1) the average fold change across all comparisons was greater than or equal to 2-fold and (2) the change in transcript level was considered significantly increased or decreased by the Affymetrix difference call metric in each microarray comparison performed. When the microarray data between the two culture conditions were compared, we found that 183 gene transcripts significantly decreased and 255 significantly increased in HFK cultured on plastic alone as compared with HFK co-cultured with feeder cells. Functional classification of a subset of these genes was performed as described in the Materials and Methods.

Many of the genes expressed at lower levels in HFK cultured on plastic alone as compared with HFK in co-culture with feeder cells are characteristically expressed and involved in keratinocyte differentiation. Many of these genes are involved in formation of the cornified envelope, a highly specialized proteinaceous structure present on the surface of the epidermis (Kalinin et al, 2002) (Table I). We also found several known components of keratinocyte desmosomes downregulated in HFK cultured on plastic alone (Kitajima, 2002). In addition, several other genes which code for proteins specifically expressed in differentiating keratinocytes were found to be downregulated in HFK cultured on plastic alone, including SPINK5, lymphocyte antigen 6 complex (E48), interleukin-1 receptor antagonist, and fatty acid binding protein 5 (Brakenhoff et al, 1995; Corradi et al, 1995; Olsen et al, 1995; Komatsu et al, 2002). Expression of keratin intermediate filaments is known to be altered during the process of differentiation. Keratins such as 1, 10, 4, and 13 are preferentially expressed in suprabasal, differentiating keratinocytes in an epidermis (Dale et al, 1990) and were also found to be downregulated in HFK cultured on plastic alone compared with HFK co-cultured with feeder cells.

Table I.

Functional classification of differentiation-associated genes downregulated in normal human foreskin keratinocytes grown on tissue culture plastic alone

Gene description Gene ID UniGene ID Fold
reduction
Cornified envelope formation
  Small proline-rich protein 3 NM_005416 Hs.139322 18.0
  Kallikrein 7 (stratum corneum chymotryptic enzyme) NM_005046 Hs.151254 17.7
  Transglutaminase 1 (keratinocyte transglutaminase) NM_000359 Hs.22 14.6
  Small proline-rich protein 1A NM_005987 Hs.211913  9.1
  Sciellin NM_003843 Hs.115166  8.6
  Involucrin NM_005547 Hs.157091  8.4
  Envoplakin NM_001988 Hs.25482  8.4
  S100 calcium-binding protein A9 NM_002965 Hs.112405  5.4
  Elafin/SKALP NM_002638 Hs.112341  5.2
  Periplakin NM_002705 Hs.74304  4.6
  Small proline-rich protein 1B NM_003125 Hs.1076  4.5
  Small proline-rich protein 2B NM_006945 Hs.231622  4.0
  Cystatin A NM_005213 Hs.2621  2.7
Desmosomal proteins
  Desmoglein 1 NM_001942 Hs.2633 32.0
  Plakophilin 1 NM_000299 Hs.198382  4.8
  Desmocollin 1 NM_004948 Hs.69752  3.5
  Plakophilin 3 NM_007183 Hs.26557  2.2
Keratins
  Keratin 4 X07695 Hs.3235 >100
  Keratin 13 NM_002274 Hs.74070 58.9
  Keratin 1 NM_006121 Hs.80828  6.4
  Keratin 10 X14487 Hs.99936  4.2
Other genes downregulated
  Serine protease inhibitor, Kazal type, 5 (SPINK5) NM_006846 Hs.331555 24.0
  Lymphocyte antigen 6 complex, locus D (E48) NM_003695 Hs.3185 23.2
  Interleukin-1 receptor antagonist homolog 1 AF216693 Hs.207224  6.7
  Fatty acid binding protein 5 NM_001444 Hs.153179  6.0
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In the absence of feeder cells, HFK upregulated genes that play a role in keratinocyte migration. Many of the genes upregulated are required to produce the provisional extracellular matrix (ECM) and specific integrins needed for migration (Table II). In addition to the ECM genes themselves, several genes that code for ECM modifying proteins were also upregulated, including matrix metalloproteinases (MMP), regulators of plasmin activation, transglutaminase 2 (tissue transglutaminase), and thrombospondin 1. Since keratinocyte migration is induced during the wound healing response, it was not surprising to see several genes involved in wound healing upregulated in HFK cultured on plastic alone compared with in co-culture with feeder cells. The antagonistic TGF-β (Tumor Growth Factor β) family proteins activin βA and follistatin are known to be expressed by keratinocytes in wounded epithelium in vivo and were upregulated in HFK grown on plastic alone, as were two members of the CCN (CYR61, CTGF, NOV) family of growth and angiogenic regulators: connective tissue growth factor and cysteine-rich, angiogenic inducer 61 (Igarashi et al, 1993; Chen et al, 2001; Wankell et al, 2003). Also consistent with the observation that HFK cultured on plastic alone altered their normal program of differentiation was the increase in expression of keratins typically found in simple, non-stratified epithelia (keratins 8, 18, and 19).

Table II.

Functional classification of migration-associated genes upregulated in normal human foreskin keratinocytes grown on tissue culture plastic alone

Gene description Gene ID UniGene ID Fold
reduction
Extracellular matrix proteins
  Fibronectin 1 X02761 Hs.287820 38.1
  Chondroitin sulfate proteoglycan 2 (Versican) D32039 Hs.81800  9.4
  Collagen, type VIII, α1 NM_001850 Hs.114599  6.4
  Laminin, γ2 NM_005562 Hs.54451  4.6
  Laminin, α3 NM_000227 Hs.83450  4.3
  Laminin, β3 NM_000228 Hs.75517  3.5
  Integrin, β6 NM_000888 Hs.123125  3.3
  Transforming growth factor, β-induced, 68 kDa NM_000358 Hs.118787  2.9
  Integrin, α2 NM_002203 Hs.271986  2.4
  Integrin, αV NM_002210 Hs.295726  2.2
  Hexabrachion (Tenascin C) NM_002160 Hs.289114  2.1
  Collagen, type XVI, α1 NM_001856 Hs.26208  2.1
Matrix metalloproteinases
  Matrix metalloproteinase 10 (stromelysin 2) NM_002425 Hs.2258  7.1
  Matrix metalloproteinase 1 (interstitial collagenase) NM_002421 Hs.83169  3.9
  Matrix metalloproteinase 2 (gelatinase A) NM_004530 Hs.111301  3.2
  Matrix metalloproteinase 9 (gelatinase B) NM_004994 Hs.151738  2.3
Plasminogen activation components
  Plasminogen activator, urokinase NM_002658 Hs.77274  7.9
  Urokinase-type plasminogen activator receptor U08839 Hs.179657  3.1
  Plasminogen activator inhibitor type 1 NM_000602 Hs.82085  2.8
Keratins
  Keratin 19 NM_002276 Hs.182265  5.6
  Keratin 8 NM_002273 Hs.242463  3.9
  Keratin 18 NM_000224 Hs.65114  3.6
Other genes upregulated
  Inhibin, β A M13436 Hs.727 59.5
  Transglutaminase 2 (tissue transglutaminase) M98478 Hs.8265 24.7
  Vimentin NM_003380 Hs.297753  6.9
  Thrombospondin 1 NM_003246 Hs.87409  6.3
  Connective tissue growth factor M92934 Hs.75511  6.0
  Cysteine-rich, angiogenic inducer, 61 NM_001554 Hs.8867  3.5
  Follistatin NM_013409 Hs.9914  2.4
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Differential gene expression and phenotypic differences (differentiation in the co-culture environment versus migration in the plastic-alone culture condition), as ascertained by microarray analysis, were validated by semi-quantitative RT-PCR and immunoblot analysis. Early-passage HFK were cultured under both conditions and transcript levels for uPAR, MMP-10, integrin α2, SPRR2B and 1A, kallikrein 7, and envoplakin were analyzed. We found the expression patterns seen in the RT-PCR analysis were consistent with the microarray data (Fig 2A). Protein levels of fibronectin, laminin γ2 (a component of laminin 5), vimentin, and keratins 8, 13, and 19 were examined by immunoblot analysis. Keratin 14 was examined as an additional control since, like actin, it was not found to be differentially expressed by microarray analysis. The protein expression patterns of these genes were in agreement with the microarray data (Fig 2B). Overall, we found a high degree of correlation between our microarray results and alternative expression assays, indicating that HFK cultured in the absence of feeder cells alter their normal gene expression program of differentiation to one characteristic of keratinocyte migration.

Figure 2.

Figure 2

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Validation of microarray results. (A) Semi-quantitative RT-PCR analysis of differentially expressed gene transcripts in early passage (population doubling ∼ 6–7) Strain 1 human foreskin keratinocytes (HFK) grown under both culture conditions. The acidic ribosomal protein P0 gene, 36B4, a housekeeping gene, is included as an internal control. Transcript levels of p16 in both culture conditions are included for comparison. (B) Immunoblot analysis of differentially expressed genes in Strain 1 HFK cultured on plastic alone (lanes 1–4) and in co-culture with feeder cells (lanes 5–8). Protein levels of actin are included as a loading control. Approximate population doublings are represented below each lane.

Induction of p16 expression correlates with markers of keratinocyte migration

FAK has been shown to be important in cellular spreading and migration through mediating integrin-dependent signaling (Gates et al, 1994; Schlaepfer et al, 1999; Kim et al, 2000). For migration to occur, keratinocytes must acquire an activated phenotype that confers the ability to produce and migrate over a provisional extracellular matrix (Coulombe, 1997). It has been previously observed that FAK activity is induced in activated keratinocytes (Kim et al, 2001). Although FAK transcript was not found to be increased by microarray analysis in HFK cultured on plastic alone compared with HFK in co-culture with feeders, FAK activity is primarily manifested by tyrosine phosphorylation, not an increase in transcription (Schlaepfer et al, 1999). Based on these observations, we hypothesized that FAK may be differentially phosphorylated in the two culture conditions. FAK autophosphorylation at Tyr-397 promotes the formation of a signaling complex involved in transducing signals from the extracellular matrix to the intracellular environment and was the focus of our examination. To determine the activation status of FAK in both culture conditions, FAK phosphorylation at Tyr-397 was assayed by immunoblotting. FAK was phosphorylated at Tyr-397 almost exclusively in HFK grown on plastic alone (Fig 3A). Immunocytochemistry was performed to establish whether FAK was being recruited to focal adhesions, the site at which FAK becomes active and autophosphorylates Tyr-397. As expected, FAK was found to co-localize with vinculin, a marker of focal adhesions, in HFK cultured on plastic alone (Fig 3B). In contrast, FAK staining in HFK cultured with feeder cells was diffuse throughout the cytoplasm (data not shown). We also confirmed that HFK with increased levels of the migration markers Tyr-397 autophosphorylated FAK and uPAR, a component of the plasminogen activation system, were selectively expressing p16. We found the overall correlation for both pTyr-397 FAK/p16 and uPAR/p16 co-staining to be 90%–98% (Fig 4). HFK staining negative for FAK autophosphorylation or uPAR showed little or no increase in p16 protein. Thus, the induction of p16 expression in HFK cultured on plastic alone is tightly correlated with markers of migration.

Figure 3.

Figure 3

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Activation of focal ahesion kinase (FAK) in keratinocytes cultured on plastic alone. (A) Immunoblot of both pTyr397-FAK and total FAK in Strain 3 human foreskin keratinocytes (HFK) cultured on plastic alone (lanes 1–4) and in co-culture with feeder cells (lanes 5–9). Protein levels of actin are included as a loading control. Approximate population doublings are represented below each lane. (B) Immunocytochemical staining for both vinculin and FAK in early passage (population doubling ∼ 6–7) Strain 2 HFK grown on plastic alone. Arrows indicate positive staining for vinculin and FAK in focal adhesions. Magnification = × 600. Scale bar = 25 micrometers.

Figure 4.

Figure 4

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Co-staining of autophosphorylated focal ahesion kinase (FAK) and urokinase plasminogen activator receptor (uPAR) with p16. Mid- to late-passage (population doubling ∼ 12–17) Strain 1 human foreskin keratinocytes (HFK) cultured on plastic alone were stained for uPAR and p16 or pTyr-397 phosphorylated FAK and p16. DAPI staining was performed for visualization of HFK nuclei. Magnification = × 400. Scale bar = 25 micrometers.

Inhibition of tyrosine kinase activity or uPA/uPAR function reduces p16 expression

As is the case in FAK activation, tyrosine phosphorylation has been found to play a significant role in both integrin signaling and cell migration (Danen et al, 1998; Longhurst and Jennings, 1998; Schlaepfer et al, 1999). In addition to tyrosine kinase activity, the urokinase plasminogen activation components uPA and its receptor, uPAR, have also been shown to serve many diverse functions in cell migration (Blasi and Carmeliet, 2002). Based on these observations, and our findings that both FAK phosphorylation status and uPA/uPAR expression were differentially modulated in the two culture conditions, we hypothesized that p16 was being induced during migration by a signaling pathway or pathways that utilized either tyrosine phosphorylation and/or uPA/uPAR interactions. Herbimycin A, a non-specific inhibitor of tyrosine kinases, and Amiloride, a competitive inhibitor of uPA, have both been shown to inhibit keratinocyte migration in vitro (Kim et al, 2001; Daniel and Groves, 2002) and were used in our studies to inhibit migration of HFK cultured on plastic alone. Using an outgrowth migration assay, we treated primary HFK cultured on plastic alone with different concentrations of either Herbimycin A or Amiloride and assayed for keratinocyte migration and p16 expression. We found that both Herbimycin A and Amiloride significantly inhibited HFK migration in the outgrowth assay (p = 0.031 for 219 nM Herbimycin A, p = 0.006 for 875 nM Herbimycin A, and p = 0.022 for 50 μM Amiloride) (Figs 5A and 6A). Morphologically, HFK treated with either Herbimycin A or Amiloride, in either the outgrowth migration assay or when plated at low density on plastic alone, displayed more sheet-like growth with cells closely packed together. In contrast, untreated, control cultures displayed many cells individually migrating outward from the preformed circular keratinocyte sheet (Figure S1). Furthermore, it was observed that both Herbimycin A and Amiloride treatment reduced p16 expression at both the mRNA and protein levels (Figs 5B, C and 6B, C). Consistent with its role as a tyrosine kinase inhibitor, Herbimycin A also reduced the levels of autophosphorylated FAK (Fig 5B). Real-time RT-PCR analysis confirmed that p16 mRNA levels were significantly different between untreated HFK and those treated with either Herbimycin A or Amiloride, although there was no significant difference in p16 mRNA levels between the two Herbimycin concentrations examined (Fig 5D). Thus, inhibition of tyrosine kinase activity or uPA/uPAR function reduces keratinocyte migration and p16 expression in HFK cultured on plastic alone.

Figure 5.

Figure 5

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Herbimycin A inhibits keratinocyte migration and p16 expression. (A) Early-passage (population doubling ∼ 5–7) Strain 3 human foreskin keratinocytes (HFK) were grown in an outgrowth migration assay on plastic alone for 7 d with or without Herbimycin A. Error bars represent standard deviation of at least three separate circular monolayer sheets. (B) Immunoblot of pTyr397-FAK (focal ahesion kinase), total FAK, and p16 in Strain 3 HFK treated with Herbimycin A. Protein levels of actin are included as a loading control. Herbimycin A concentrations used are represented below each lane. (C) Semi-quantitative RT-PCR analysis of p16 in Strain 3 HFK treated with Herbimycin A. GAPDH, a housekeeping gene, is included as an internal control. Herbimycin A concentrations used are represented below each lane. (D) Real-time RT-PCR analysis of p16 in Strain 3 HFK treated with Herbimycin A. Levels of p16 mRNA were standardized against expression levels of the 18S rRNA gene transcript and then normalized relative to the untreated, control group. Error bars represent standard error of the mean.

Figure 6.

Figure 6

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Amiloride inhibits keratinocyte migration and p16 expression. (A) Early-passage (population doubling ∼ 5–7) Strain 3 human foreskin keratinocytes (HFK) were grown in an outgrowth migration assay on plastic alone for 7 d with or without Amiloride. Error bars represent the standard deviation of at least three separate circular monolayer sheets. Amiloride concentrations 250 μM and above were found to be cytotoxic to HFK and cells treated with these concentrations were not used for further analysis. (B) Immunoblot of p16 in Strain 3 HFK treated with Amiloride. Protein levels of actin are included as a loading control. The Amiloride concentration used is represented below lane 2. (C) Semi-quantitative RT-PCR analysis of p16 in Strain 3 HFK treated with Amiloride. GAPDH, a housekeeping gene, is included as an internal control. The Amiloride concentration used is represented below lane 2. (D) Real-time RT-PCR analysis of p16 in Strain 3 HFK treated with Amiloride. Levels of p16 mRNA were standardized against expression levels of the 18S rRNA gene transcript and then normalized relative to the untreated, control group. Error bars represent standard error of the mean.

Discussion

It is known that human keratinocytes cultured under different conditions exhibit differences in both replicative capacity and p16 expression. In this study, we have shown that HFK cultured on plastic alone exhibit a phenotype characteristic of a keratinocyte migration response and that p16 expression is induced under these conditions by a mechanism involving tyrosine kinase activity and the urokinase plasminogen activation system. This phenotypic change in keratinocyte behavior and gene expression suggests an identity for the culture-induced stress experienced when keratinocytes are grown in the absence of post-mitotic feeder cells, and poses new questions as to a possible role for p16 in keratinocyte migration.

Growth arrest and p16 expression in different culture conditions

Similar to what has been observed previously, we found that both replicative capacity and p16 expression are altered in human keratinocytes grown under two different culture conditions. Our results are consistent with previous reports that have shown a decrease in p16 expression and increase in proliferative capacity in the co-culture system; however, unlike previous reports that characterized the increase in cellular lifespan to be approximately 20–30 PD, we found it to be more modest (Ramirez et al, 2001; Rheinwald et al, 2002; Fu et al, 2003; Kang et al, 2003, 2004). What accounts for the variation seen between co-culture results remains to be identified; however, possibilities include differences in precise media constituents, passage protocols, feeder cell number, and/or the method of induction of the feeder cell post-mitotic state.

It is of interest that others have also reported that p16 protein levels ultimately increase as human keratinocytes age in the co-culture environment (Rheinwald et al, 2002; Fu et al, 2003; Kang et al, 2004). This observation indicates that, although p16 is induced to a greater extent in the absence of feeder cells, there is still some mechanism that eventually causes an increase in p16 expression in the co-culture environment. This may be through a variety of processes including exact culture conditions and/or induction of p16 through telomere loss or damage as has been recently reported for human fibroblasts (Jacobs and de Lange, 2004).

p16 expression is co-regulated with keratinocyte migration genes by a mechanism involving tyrosine kinase activity and uPA/uPAR function

The stimulus for p16 induction under certain culture conditions has not been fully elucidated; however, it has been proposed that cellular stress is responsible for this change in gene expression (Sherr and DePinho, 2000; Shay and Wright, 2001; Lowe and Sherr, 2003; Ben-Porath and Weinberg, 2004). During the wound healing process, keratinocytes undergo a complex reprogramming of gene expression that allows them to alter their normal behavior of terminal differentiation to one of migration and proliferation. This change induces a switching of keratinocyte integrin profiles from one that favors attachment to the basal lamina (mediated by α6β4 integrins) to one necessary for migration over a provisional matrix (α5β1, αVβ6, αVβ5, α3β1, and α2β1) (Martin, 1997; Nguyen et al, 2000). To facilitate migration, keratinocytes must also acquire the ability to remodel ECM proteins. Activation of plasmin, the main fibrinolytic enzyme necessary to dissolve a fibrin clot, is achieved by either tissue-type plasminogen activator (tPA) or uPA. To increase the likelihood of plasmin activation, uPAR is also known to be upregulated during the wound healing process (Blasi and Carmeliet, 2002). Keratinocyte migration is also aided by upregulation of MMP-1, -9, and -10 (Martin, 1997). Additionally, in an effort to both restore the basal lamina and aid migration, keratinocytes engaged in the wound healing response produce extracellular matrix proteins including laminin, fibronectin, and collagen (O'Toole, 2001; Kirfel et al, 2003).

Our microarray results and subsequent validation indicate that, in contrast to differentiating HFK in co-culture with feeder cells, HFK cultured on plastic alone are engaged in a migratory response that displays upregulation of several genes also involved in the wound healing process. Most of the genes mentioned above were found to be upregulated in HFK grown on plastic alone as were components of the TGF-β signaling pathway, also thought to play a role in migration during the keratinocyte wound healing response (Martin, 1997; Decline et al, 2003; Wankell et al, 2003). From the noticeable differences in culture conditions, it is probable that this change in phenotype is induced by a lack of specific cell–cell contacts between keratinocytes and/or fibroblasts. In an effort to exclude differences in culture media as the reason for the phenotypic switch, we attempted to culture HFK in E-media without feeder cells; however, HFK cultured in this fashion did not survive beyond one passage (data not shown). It should be noted that the concentration of calcium in E-media is much higher than in keratinocyte serum-free media (KSFM), and calcium has been shown to be an important determinant in causing keratinocyte differentiation (Boyce and Ham, 1983). We attempted to determine whether the increased calcium content of E-media was responsible for inhibition of p16 expression in the co-culture environment but when KFSM was supplemented with a similar level of calcium (1.28 mM), we actually observed an increase in p16 expression (data not shown). Thus, it appears that the feeder cells are a primary factor responsible for inhibition of p16 expression in the co-culture environment.

Both FAK activation and uPA/uPAR interactions have been shown to be involved in cell migration. The recruitment of FAK to focal adhesions and the consequent autophosphorylation of Tyr-397 forms a signaling complex involved in transducing extracellular signaling to the nucleus through a series of tyrosine phosphorylation events that involve signaling proteins such as p130Cas and Shc (Schlaepfer et al, 1999). Binding of pro-uPA to uPAR activates uPA, facilitating the generation of plasmin from plasminogen, resulting in proteolysis of the ECM and increased mobility of migrating cells. In addition to this proteolytic mechanism of migration, however, uPA/uPAR interactions have also been shown to be connected to a variety of intracellular signaling pathways that involve both tyrosine and serine protein kinases such as Src, Hck, FAK, and the Ras/mitogen-activated protein kinase pathway (Ras/MAPK) (Blasi and Carmeliet, 2002). Based on these observations, we focused our studies on both FAK and the urokinase plasminogen activation system. We found that the expression levels of uPA and uPAR as well as the phosphorylation status of FAK were both modulated by differences in culture conditions, and that in HFK cultured on plastic alone, the induction of p16 expression was tightly correlated with increased cellular levels of either autophosphorylated FAK or uPAR. Furthermore, when uPA/uPAR functions, or the cascade of tyrosine phosphorylation downstream of both uPA/uPAR and FAK, were inhibited we observed a reduction in both HFK migration and p16 expression.

Since FAK can be activated by uPA/uPAR interactions, and both are involved in activating signaling pathways that involve tyrosine phosphorylation, it is possible that both Herbimycin A and Amiloride were inhibiting the same pathway responsible for induction of p16 expression during keratinocyte migration. The downstream signaling pathways influenced by both FAK and uPA/uPAR are numerous and we are currently in the process of dissecting out which signaling intermediates are involved in transducing the migratory signal from uPA/uPAR and FAK to the nucleus and ultimately the p16 promoter. One such pathway, the Ras/MAPK, is intriguing as oncogenic expression of Ras has been shown to induce p16 expression and consequent growth arrest in human fibroblasts (Serrano et al, 1997; Lin et al, 1998; Ohtani et al, 2001).

In agreement with our findings that p16 induction is associated with a migratory response, it has been observed that p16 expression is induced in human keratinocytes subjected to scraping in vitro (Natarajan et al, 2003). Furthermore, it was shown by Natarajan and colleagues that p16 and laminin γ2 were co-expressed under these conditions, a result consistent with our finding that laminin γ2, as well as laminins α3 and β3, was upregulated in HFK cultured on plastic alone compared with that in co-culture with feeder cells. It has been proposed that p16 induction during migration may have evolved to facilitate wound closure by halting proliferation in favor of increased cell motility (Natarajan et al, 2003). In several contexts, however, p16 has been shown to inhibit migration of various cell types (Fahraeus and Lane, 1999; Adachi et al, 2001) and may play a role in contact-mediated growth inhibition (Wieser et al, 1999). An alternative hypothesis is that migration-induced p16 expression evolved to prevent constitutive activation of the genes necessary for migration. Thus, loss of p16 in cancer cells may allow for continued activation of migratory genes and an increased incidence of invasion. If this is the case, future therapies designed to modulate p16 expression in invasive carcinomas could prevent both tumor proliferation and metastasis. Further examination of the role that p16 may play in migration is currently in progress.

In summary, we have shown that human keratinocytes, grown in the absence of feeder cells, are subjected to an additional culture-induced stimulus that co-regulates the expression of several genes involved in migration and p16 expression by a mechanism involving tyrosine kinase activity and uPA/uPAR function. These results provide new insight into keratinocyte migration as well as p16 induction and may ultimately provide a mechanism for the cellular stasis experienced when primary keratinocytes are cultured in the absence of feeder cells.

Materials and Methods

Cell culture

HFK were isolated as previously described (Blanton et al, 1991). Foreskin tissue was obtained using a protocol approved by the University of Iowa Institutional Review Board in accordance with HIPAA guidelines and the Declaration of Helsinki Principles. HFK were grown under two different culture conditions. In the “plastic” condition, HFK were grown on tissue culture plastic in KSFM (Invitrogen, Carlsbad, California: 10724-011) supplemented with 0.2 ng per mL human recombinant epidermal growth factor (EGF), 30 μg per mL bovine pituitary extract (BPE), and 1% Penicillin–Streptomycin (Invitrogen: 15140-122). In the “feeders” condition, HFK were grown on tissue culture plastic in co-culture with post-mitotic, γ-irradiated J2 3T3 fibroblasts in E-media. E-media consist of a base of Dulbecco's modified Eagle's media (Invitrogen: 11965-092) and HAM F-12 nutrient mixture media (Invitrogen: 11765-054) at a 3:1 ratio, respectively, supplemented with 10% fetal bovine serum (FBS) (Invitrogen: 26140-079), 1% Penicillin–Streptomycin (Invitrogen: 15140-122), 1.36 ng per mL tri-iodo-thyronine (Sigma, St Louis, Missouri: T5516), 0.5 μg per mL hydro-cortisone (Sigma: H0396), 8.4 ng per mL cholera toxin (Sigma: C3012), 5.0 μg per mL transferrin (Sigma: T1147), 5.0 μg per mL insulin (Sigma: I6634), and 4.5 ng per mL EGF (Invitrogen: 13247-051). The final calcium concentrations in both KSFM and E-media were 0.09 and 1.28 mM, respectively. In the co-culture condition, irradiated fibroblasts were removed by incubation with a solution of 0.05% trypsin (Invitrogen: 15400-054), 50 mM HEPES buffer solution (Invitrogen: 15630-080) in Hanks balanced salt solution (Invitrogen: 14170-112). This protocol effectively removed the fibroblast feeder cells while leaving the keratinocytes attached. HFK were removed by further incubation in 0.05% trypsin/EDTA solution (Invitrogen: 25300–054). Trypsinized HFK were pelleted by centrifugation and replated onto plastic alone or into co-culture with a new population of irradiated J2 3T3 fibroblasts. All cells were passaged at a 1:4 or 1:6 split ratio when 70%–80% confluent and maintained at 37°C with 5% CO2. PD were calculated using the following equation: PDn = PD(n−1) + log10[split ratio]/log10[2] (Reznikoff et al, 1987).

RNA and protein isolation

Both RNA and protein were collected at selected PD when HFK reached 70%–80% confluence. RNA and protein were collected from HFK grown with feeders after the removal of the irradiated fibroblasts. Total RNA was collected using TriReagent (Molecular Research Center, Cincinnati, Ohio) and purified according to the manufacturer's instructions. Total protein was collected using WE16 lysis buffer, as previously described (Foster and Galloway, 1996) and quantified using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, California) according to the manufacturer's instructions.

Oligonucleotide microarray analysis

RNA samples were prepared for hybridization as previously described (Duffy et al, 2003). Affymetrix HG-U133A GeneChips were hybridized, washed, and stained by the University of Iowa DNA Facility according to Affymetrix protocols (Affymetrix, Santa Clara, California). Oligonucleotide arrays were scanned by the University of Iowa DNA Facility using a confocal scanner manufactured for Affymetrix by Molecular Dynamics (Sunnyvale, California). Data analysis was performed using the GeneChip software (Version 5.0) supplied with the Affymetrix instrumentation system. RNA was collected from two independent strains of primary HFK (Strain 1 and Strain 2) serially subcultured under both culture conditions to approximately half of their maximum lifespan (Strain 1: PD ∼ 8 and Strain 2: PD ∼ 14). For each condition, one GeneChip was analyzed and the following comparisons were made: Strain 1 on plastic versus Strain 1 on feeders, Strain 1 on plastic versus Strain 2 on feeders, Strain 2 on plastic versus Strain 1 on feeders, and Strain 2 on plastic versus Strain 2 on feeders. For each comparison, the co-culture condition was used as a baseline. For each gene, across all four GeneChip comparisons, the average fold change was calculated from the signal log ratio value. The change in gene expression level was considered significant if (1) the average fold change was greater than or equal to 2-fold and (2) the change in transcript level was considered significant by the Affymetrix difference call metric (only values of I = Increase and D = Decrease were accepted) in each comparison performed. Functional classification of a subset of these genes was performed by referencing public databases such as Netaffx (http://www.affymetrix.com/analysis/index.affx) and utilizing microarray analysis software including D-Chip (Harvard, Boston, Massachusetts) and GeneSpring (Silicon Genetics, Redwood City, California).

Immunoblot analysis

Immunoblot analyses of cellular protein levels were performed as previously described (Foster et al, 1994) using 20 μg of protein lysate, run on polyacrylamide gels of the appropriate concentration, and transferred onto Immobilon-P membranes (Millipore, Billerica, Massachusetts). Membranes were stripped by incubation and agitation at 50°C in a solution of 100 mM 2-mercaptoethanol, 2% (wt/vol) SDS, 62.5 mM Tris-HCl at pH 6.7. Stripped membranes were then reblocked and reprobed with different antibodies. The following antibodies were used in this study: p16 (Pharmingen, San Diego, California: G175-405), Fibronectin (BD Transduction Laboratories, San Jose, California: 10), Laminin γ2 (Chemicon International, Temecula, California: D4B5), Vimentin (Santa Cruz Biotechnology, Santa Cruz, California: V9), Keratin 19 (ICN Biomedicals, Irvine, California: KS19.1), Keratin 14 (Cymbus Biotechnology, Chandlers Ford, UK: LL002), Keratin 13 (Santa Cruz Biotechnology: DE-K13), Keratin 8 (Sigma: K8.60), pY397-FAK (BD Transduction Laboratories: 14), FAK (Upstate Cell Signaling Solutions, Charlottesville, Virginia: 06-543), and Actin (Santa Cruz Biotechnology.: I-19). Blots were probed with the appropriate HRP-conjugated secondary antibody: goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania), goat anti-rabbit IgG (Santa Cruz Biotechnology), or donkey anti-goat IgG (Santa Cruz Biotechnology). Detection was performed using the Western Lightning chemiluminescence kit (Perkin-Elmer Life Sciences, Boston, Massachusetts).

Semi-quantitative RT-PCR analysis

cDNA synthesis reactions were carried out using the RETROscript kit (Ambion, Austin, Texas). RNA samples (2 μg) were used as the template for single-stranded cDNA synthesis reactions primed with the oligo dT primers included in the RETROscript kit. PCR analysis was carried out using the SuperTaq kit (Ambion) according to the manufacturer's instructions. RT-PCR reaction products were separated by agarose gel electrophoresis, stained with ethidium bromide, and visualized by UV light. Primer sequences and PCR conditions used for semi-quantitative RT-PCR amplification of the indicated genes are provided as Supplemental Materials (Table S1).

Real-time RT-PCR analysis

cDNA synthesis reactions were carried out as described above using the RETROscript kit. Real-time PCR was performed on 100 ng of cDNA using the Sybr Green system (Applied Biosystems, Foster City, California, Product Number: 4309155) and a 7000 Sequence Detection System real-time thermalcycler (Applied Biosystems). Real-time PCR reactions were carried out for both p16 and 18S (a ribosomal gene analyzed as a control) at an annealing temperature of 60°C for 40 cycles. Analysis was performed using the ABI Prism 7000 SDS software supplied with the 7000 Sequence Detection instrumentation system. Levels of p16 were standardized against 18S levels and normalized by relative comparison. All real-time reactions were carried out in triplicate, and error bars were created using standard error of the mean.

Immunocytochemistry

Vinculin and FAK Early-passage HFK (PD ∼ 5–7) were cultured on glass coverslips without feeder cells in KSFM. Cells were fixed with 3.7% formaldehyde in PBS for 10 min at room temperature. Cells were permeabilized with 0.5% Triton X-100 in PBS for 7 min. Coverslips were blocked in 5% normal donkey serum, 5% normal goat serum, 1% BSA, and 0.1% Tween-20 in PBS for 45 min. Coverslips were incubated for 1 h with a mixture of both vinculin (antibody 7F9, a gift from K. Burridge, University of North Carolina) and FAK (Upstate Cell Signaling Solutions: 06-543) primary antibodies. FAK primary antibody was diluted to a ratio of 1:100 in the provided solution of vinculin antibody. A mixture of donkey anti-mouse Texas-red conjugated (Jackson ImmunoResearch Laboratories) and goat anti-rabbit fluorescein-conjugated (Chemicon International) secondary antibodies, both diluted 1:100 in blocking buffer, was incubated with the coverslips for 1 h. The coverslips were mounted on glass slides using a 60% glycerol solution. Images were collected on a Bio-Rad Radiance 2100 MP Confocal/Multiphoton microscope (Bio-Rad Laboratories), at the University of Iowa Central Microscopy Core Facility. Images were analyzed and merged using ImageJ software (National Institutes of Health). PBS washes were performed between each step in all immunocytochemistry protocols.

pTyr-397 FAK, uPAR, and p16 Mid- to late-passage HFK (PD ∼ 12–17) were grown on permanox chamber slides without feeder cells in KSFM. Cells were fixed with 3.7% formaldehyde in PBS for 10 min at room temperature. Cells were permeabilized with 0.5% Triton X-100 in PBS for 7 min. Slides were blocked in 10% normal donkey serum, 1% BSA in PBS for 45 min. Slides were incubated with pTyr-397 FAK (BD Transduction Laboratories: 14) or uPAR (American Diagnostica, Greenwich, Connecticut: 3936) primary antibodies both diluted in blocking buffer at a ratio of 1:100 for 1 h. Texas-red conjugated donkey anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories), diluted 1:100 in blocking buffer, was incubated with the chamber slides for 1 h. Slides were then incubated with an Oregon-green-conjugated p16 primary antibody diluted 1:40 in a blocking buffer consisting of 10% normal goat serum, 1% BSA in PBS for 1 h. The Oregon-green conjugated p16 antibody was created using a primary antibody to p16 (Pharmingen: G175-405) and a Zenon Oregon-green labeling kit (Molecular Probes, Eugene, Oregon) according to the manufacturer's instructions. Chamber slides were mounted with VECTA-SHIELD mounting medium with DAPI (Vector Laboratories, Burlingame, California) to provide for visualization of HFK nuclei. Images were collected on a Nikon Eclipse E800 fluorescence microscope (Nikon Corporation, Tokyo, Japan). To ascertain pTyr-397 FAK/p16 and uPAR/p16 co-staining, HFK staining positive for the different antibodies were visualized and quantified in four separate microscopic fields.

Migration assays

Outgrowth migration assays were performed as previously described (Ura et al, 2004). Briefly, early-passage HFK (PD ∼ 5–7) were suspended in KSFM or KSFM containing different concentrations of either Herbimycin A (Sigma: H6649) or Amiloride (Sigma: A7410). Approximately 4 × 104 HFK were then plated inside a 5 mm cloning ring placed on a non-coated, plastic tissue culture plate and allowed to attach for 4 h. After the HFK had attached, the cloning ring was removed to yield a circular monolayer sheet. Outward migration was measured for 7 days with media changes every other day. In treatment groups, Herbimycin A or Amiloride was maintained in the media for the duration of the experiment. After 7 d, protein and RNA were collected as described above. For each treatment group, triplicate monolayer sheets were plated, measured for migration, and collected for protein and RNA. Statistical significance was measured by a paired Student's t test.

Supplementary Material

supfig
suptbl

Acknowledgments

We thank Keith Burridge for providing the vinculin antibody, as well as Alberto Gasparoni, Rebecca Zaharias, Gail Kurriger, Jim Martin, Martine Dunnwald, Jackie Bickenbach, Rich Seftor, and the Rick Domann laboratory for help with immunocytochemistry protocols, immunoblot components, and real-time PCR analysis. We thank Kevin Ault for procurement of foreskin tissue. We are grateful to the other members of the Klingelhutz laboratory for helpful discussions. This work was supported by a grant from the National Institute on Aging (NIA), R01 AG18265, and an American Cancer Society institutional research seed grant. Benjamin Darbro was supported by training grants from the National Heart, Lung, and Blood Institute (NHLBI), T32 HL07638, and the University of Iowa Medical Scientist Training Program (MSTP), T32 GM07337.

Abbreviations

FAK

focal adhesion kinase

HFK

human foreskin keratinocyte

PD

population doublings

Rb

retinoblastoma protein

uPAR

urokinase plasminogen activator receptor

Footnotes

Supplementary Material

The following material is available online for this article.

Figure S1 Herbimycin A and Amiloride treatment alter HFK morphology and migration patterns.

Table S1 Primer sequences and PCR conditions used for semi-quantitative RT-PCR amplification of the indicated genes.

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Supplementary Materials

supfig
NIHMS22112-supplement-supfig.pdf (83.3KB, pdf)
suptbl
NIHMS22112-supplement-suptbl.pdf (41KB, pdf)

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