SERPINE1 (PAI-1) Is a Prominent Member of the Early G₀→G₁ Transition “Wound Repair” Transcriptome in p53 Mutant Human Keratinocytes

TO THE EDITOR

Serum-stimulation of quiescent (G₀) keratinocytes initiates a temporally regulated program of transcriptional activity required for G₀/G₁ transit and subsequent entry into the proliferative cycle (Qi and Higgins, 2003). Expression profiling of such “activated” keratinocytes identified physiologically relevant subsets of cell cycle/growth state-regulated genes (Gromov et al., 2002; Gazel et al., 2003). Indeed, non-cycling human (HaCaT) keratinocytes express a differentiated (i.e., super-basal) genetic signature, whereas the serum-stimulated transcriptome approximates that of transient amplifying cells (Pivarcsi et al., 2001; Lemaitre et al., 2004). Clearly, the associated transcriptional responses dictate epidermal cell lineage commitments by impacting the expression of pathway-relevant genes (Banno et al., 2004; Lemaitre et al., 2004).

This report provides early evidence regarding the comprehensive inventory of genes expressed by human HaCaT-II4 keratinocytes during the initial stage of cell-cycle re-entry. Re-introduction of serum to quiescent HaCaT-II4 cells stimulates G₀ exit and residence in a short-lived “activated G₀ substate” (i.e., the kinetically defined G₀→G₁ transition state) (Qi et al., 2006). Microarray analysis of quiescent and 2 hours fetal bovine serum (FBS)-“ activated” HaCaT-II4 cells defined the transcriptional signature of this early G₀→G₁ window. A total of 54,675 expressed sequence-tagged genes were analyzed with 41,083 directly compared for groups A (quiescent) and B (2 hours FBS-stimulated) and a total of 35,991 reproducibly assessed for all three experimental conditions (i.e., 66% of the total sequences available; this includes group C [FBS for 2 hours in the presence of puromycin included as a first approximation of the immediate-early response cluster]). Genes exhibiting statistically significant (analysis of variance) changes (two-fold increase or decrease) distributed as follows: 1151 for A versus B, 1241 for A versus C, and 1319 for B versus C. Among the most prominently upregulated mRNA transcripts were those encoding proteins involved in the initial growth response (EGR1-4), extracellular matrix remodeling and tissue invasion (uPA, uPAR, tPA, SERPINE1 (PAI-1), PAI-2, MMP-2, MMP- 12, CYR61), transcription (Myc, Fos, Jun, KLF4, AT3, p300/pTAF), signal transduction (DUSP1, -4, -8, -10, MAPK3, TGF-α), proliferation (GADD45a, GADD45b, CDK7, cyclins), and apoptosis (CASP9, MCL2) (e.g., Figure 1a). Reverse transcription-PCR and northern blotting validated the expression data for selected genes. When more stringent criteria were applied to data filtering (i.e., set threshold of ≥ 10-fold increase), 79 genes were identified (Table 1) of which 75 also partitioned to the puromycin-resistant subset. Rank order analysis indicated that PAI-1 (SERPINE1) and the uPA receptor (PLAUR) were the most significantly elevated transcripts. uPA increased (by 12-fold) as well (by microarray and northern analyses), although maximal uPA expression occurred several hours later than PAI-1. Additional genes that comprise the “tissue repair” subset and that were upregulated > 10-fold within the first 2 hours included DTR, IL8, EREG, HBEGF, IL11, CTGF, LIF, IL6, IL1A, HAS3, SERPINB1, and TGFA. Northern blotting confirmed that PAI-1 transcripts were low to undetectable in quiescent HaCaT-II4 cells, peaked in puromycin-resistant manner 2 hours after serum addition (during residence in the initial activated G₀ substate; Qi et al., 2006), and then rapidly decreased (Figure 1b and c). Expression required EGFR/MEK/rho-ROCK signaling during the G₀→G₁ transition (Figure 1d and e). Actinomycin chase/mRNA decay and temporal assessments of mRNA abundance indicated, moreover, that PAI-1 transcripts were substantially reduced (from a maximum at 2 hours) as early as 4 hours post-stimulation decreasing further by 6–8 hours post-stimulation (i.e., approximately mid-G₁) likely due to E2F1-mediated suppression (Koziczak et al., 2001), consistent with a narrow window of serum-initiated transcription and short mRNA half-life (1.5–2 hours) (Mu et al., 1998; White et al., 2000).

Figure 1. A significant fraction of FBS-induced transcripts encode proteins involved in cell proliferation, transcriptional reprogramming, and control of pericellular proteolysis.

The clustergram plot (a) illustrates functional groupings for several highly expressed (≥ 10-fold; red to orange shading) relative to more moderately stimulated (> 2- to < 10-fold; light to dark pink colored) genes (mapped using Ingenuity Pathways software). (b, c) PAI-1 transcripts (both the 3.0 and 2.2 kb species) are low to undetectable in quiescent (Q) HaCaT-II4 keratinocytes and induced within 2 hours of serum re-introduction (FBS-2 hours) or EGF, but not by replacement with serum-free medium (medium Δ). (b, c) PAI-1 expression is effectively inhibited by prior incubation with actinomycin D (Act D) or the MEK inhibitor PD98059 (PD) but not by puromycin (Puro). (b) PAI-1 mRNA decay rates in cultures treated with Act D 2 hours after FBS addition were virtually identical to control mRNA decay profiles suggesting that PAI-1 repression was initiated between 2 and 4 hours post-serum stimulation. (d, e) Y-27632 and U0126 inhibited PAI-1 induction implicating both the rho GTPase effector ROCK and MEK, respectively, in gene control. (d) Pretreatment of quiescent cells with the EGFR inhibitor AG1478 similarly blocked PAI-1 expression indicating that EGFR ligands were major contributors to the serum-responsiveness of the PAI-1 gene. Glyceraldehyde-3-phosphate dehydrogenase (GAPD) hybridization provided a normalizing signal for (b and c) northern analysis (Mu et al., 1998; Qi et al., 2006 for details), whereas (d, e) western blots were stripped and re-probed with antibodies to ERK2 to confirm protein loading levels (described in Providence and Higgins, 2004).

Table 1.

Genes exhibiting a ≥ 10-fold increase in expression 2 hours after serum stimulation of quiescent HaCaT-II4 cells

Gene symbol	Fold-increased expression	Description
SERPINE1	97.7	Plasminogen activator inhibitor type-1
PLAUR	76.6	Urokinase plasminogen activator receptor
PLAUR	70.3	Urokinase plasminogen activator receptor
DTR	67.9	Heparin-binding epidermal growth factor-like precursor
NR4A3	65.7	Nuclear receptor subfamily 4, group A, member 3
CLC	62.0	Cardiotrophin-like cytokine
C8FW	61.1	Phosphoprotein regulated by mitogenic pathways
IL8	56.3	Interleukin 8
CLDN4	49.5	Claudin 4
GEM	48.5	GTP-binding protein overexpressed in skeletal muscle
EREG	48.0	Epiregulin
SPRR2B	46.7	Small proline-rich protein 2B
EGR2	46.3	Early growth response 2 (Krox-20 homolog)
HB-EGF	41.3	Heparin-binding epidermal growth factor
PHLDA1	40.0	Pleckstrin homology-like domain, family A, member 1
IL11	38.1	Interleukin 11
CTGF	37.8	Connective tissue growth factor
KRTAP3-1	37.4	Keratin-associated protein 3-1
LIF	36.9	Leukemia inhibitory factor
EDN1	36.6	Endothelin 1
FOSL1	35.8	FOS-like antigen 1
TNFAIP3	34.4	Tumor necrosis factor, α-induced protein 3
TNFAIP3	32.9	Tumor necrosis factor, α-induced protein 3
EGR3	32.9	Early growth response 3
PTGS2	32.5	Prostaglandin-endoperoxide synthase 2
COPEB	31.1	Core promoter element-binding protein
ZFP36	30.6	Zinc finger protein 36
APOBEC3A	30.2	Apolipoprotein B mRNA editing enzyme
COPEB	28.9	Core promoter element-binding protein
DUSPI	28.3	Dual specificity phosphatase 1
NR4A2	28.0	Nuclear receptor subfamily 4, group A, member 2
FOXA1	27.4	Forkhead box A1
EDN1;ET1	26.7	Endothelin 1
DUSP10	25.3	Dual specificity phosphatase 10
IL6	25.1	Interleukin 6
SOCS3	24.8	Suppressor of cytokine signaling 3
KLF4	24.1	Kruppel-like factor 4
NR4A2	23.4	Nuclear receptor subfamily 4, group A, member 2
PTGS2	23.1	Prostaglandin-endoperoxide synthase 2
C20oorf16	21.4	Chromosome 20 open reading frame 16
DUSP4	21.0	Dual specificity phosphatase 4
ATF3	19.3	Activating transcription factor 3
PIM1	19.1	Pim-1 oncogene
MAFF-like	18.6	v-maf-like
JUN	18.3	v-jun sarcoma virus 17 oncogene homolog
NR4A2	18.1	Nuclear receptor subfamily 4, group A, member 2
IL1A	18.0	Interleukin 1, alpha
GADD45B	17.4	Growth arrest and DNA-damage-inducible, beta
HAS3	17.3	Hyaluronan synthase 3
EMP1	16.8	Epithelial membrane protein 1
SLC20A1	16.8	Solute carrier family 20 (phosphate transporter), member 1
GADD45B	16.8	Growth arrest and DNA-damage-inducible, beta
DSCR1	16.7	Down syndrome critical region gene 1
GADD45B	16.6	Growth arrest and DNA-damage-inducible, beta
ARTN	16.6	Artemin
B3GNT5	16.1	UDP-GlcNAc:betaGal β-1,3-N-acetylglucosaminyltransferase 5
RGC32	16.0	RGC32 protein
JUN	15.0	v-jun sarcoma virus 17 oncogene homolog
TRIF	14.9	TIR domain containing adaptor-inducing interferon-β
KLF4	14.6	Kruppel-like factor 4
IER3	14.5	Immediate early response 3
SERPINB1	14.4	Serine (or cysteine) proteinase inhibitor, clade B, member 1
RFX2	14.4	Regulatory factor X, 2
SGK	13.5	Serum/glucocorticoid-regulated kinase
ADM	13.5	Adrenomedulin
IL1B	12.9	Interleukin 1-β
SPRR3	12.8	Small proline-rich protein 3
HSPC159	12.6	Human galectin-related protein
EMP1	12.5	Epithelial membrane protein 1
PTGER4	12.3	Prostaglandin E, receptor 4
PLEKHC1	12.2	Pleckstrin homology domain containing, family C, member 1
LM07	12.1	LIM domain only 7
PLEKHC1	11.9	Pleckstrin homology domain containing, family C, member 1
ATF3	11.7	Activating transcription factor 3
PDCD1L1	11.7	Programmed cell death 1 ligand 1
CNK	11.6	Cytokine-inducible kinase
PPP1R15A	11.5	Protein phosphatase 1, regulatory (inhibitor) subunit 15A
FST	11.5	Follistatin
MAFF	11.1	v-maf fibrosarcoma oncogene avian homolog F
IFRD1	11.0	Interferon-related developmental regulator 1
SNARK	10.9	Likely ortholog of rat SNF1/AMP-activated protein kinase
ODC1	10.7	Ornithine decarboxylase 1
SLC2A3	10.4	Solute carrier family 2 (facilitated glucose transporter), member 3
TGFA	10.4	Transforming growth factor, alpha
GADD45A	10.3	Growth arrest and DNA-damage-inducible, alpha
B3GNT5	10.2	UDP-GlcNAac:betaGal β-1,3-N-acetylglucosaminyltransferase 5
SLC2A14	10.2	Solute carrier family 2 (facilitated glucose transporter), member 14
DUSP5	10.1	Dual specificity phosphatase 5
CUL1	10.1	Cullin 1
PPP1R3C	10.0	Protein phosphatase 1, regulatory (inhibitor) subunit 3C

ANOVA, analysis of variance; FBS, fetal bovine serum; cDNA, complementary DNA. RNA isolated from quiescent or 2 hours FBS-stimulated keratinocytes was converted into single-stranded cDNA using Superscript II reverse transcriptase and the GeneChip T7 promoter primer kit. Double-stranded cDNA was prepared, biotinylated cRNA generated, hydrolyzed to 35–200 base fragments, and hybridized to the Affymetrix Human Genome U133 Plus 2.0 oligonucleotide array. Arrays were washed, stained with streptavidin-phycoerythrin, scanned and images analyzed qualitatively with Affymetrix GCOS software; probe signal outputs (pivot tables) were imported as text files into GeneSpring v6.1. Values below 0.01 were set to 0.01 and each was divided by the 50th percentile of all measurements in that sample. Individual gene data points, in each of the experimental groups, were divided by the median value in the corresponding control sets. ANOVA analysis (95% confidence) compared groupings as follows: B versus A, C versus A, and B versus C. The statistically significant genes (in triplicate assessments) were filtered to obtain lists based on expression levels (for this paper, 10× increased relative to the corresponding control). Signal reproducibility is evidenced by expression level values for duplicated genes on each array (indicated by color coding in bold font).

Activation of a wound repair transcript profile appears to be a general response to serum addition (Iyer et al., 1999; this study). The present findings indicate, furthermore, that PAI-1 is the most prominent member of the keratinocyte “serum response transcriptome”. Several SERPINS (i.e., PAI-1, protease nexin-1), in fact, modulate the complex process of injury resolution through control of focalized plasminmediated matrix remodeling, cell migration, and apoptosis (e.g., Bajou et al., 2001; Li et al., 2000; Deng et al., 2001; Degryse et al., 2004; Rossignol et al., 2004; Wang et al., 2005). Targeted PAI-1 knockdown/overexpression and protein add-back approaches, moreover, support the contention that PAI-1 participates within the global program of injury to coordinate cycles of cell-to-substrate adhesion/detachment and/or maintain a stromal “scaffold” to satisfy the prerequisites for both G₁/S transition and effective cellular migration (Planus et al., 1997; Chazaud et al., 2002; Palmeri et al., 2002; Providence et al., 2002; Czekay et al., 2003; Providence and Higgins, 2004). PAI-1 is also expressed at high levels in senescent cells where it likely interferes with uPA-dependent growth factor activation (Mu et al., 1998; Kortlever et al., 2006). Certain “senescence-associated” genes (i.e., p16^INK4a, PAI-1) may actually function in the wound repair program by inhibiting proliferation while promoting migration (Chan et al., 2001; Ploplis et al., 2004; Darbro et al., 2005; Kortlever et al., 2006; Natarajan et al., 2006). Indeed, keratinocytes at the leading edge during wound re-epithelialization are less mitotically active than cells more displaced from the motile front and express relatively high levels of PAI-1 (Garlick and Taichman, 1994; Jensen and Lavker, 1996; Providence and Higgins, 2004). Collectively, these data suggest that PAI-1 may regulate the temporal cadence of cell-cycle progression in replicatively competent cells as part of the injury repair program.

Abbreviations

FBS

fetal bovine serum