Reduced Mitochondrial Properties in Putative Progenitor/Stem Cells of Human Keratinocytes

Culture of normal human keratinocytes and adhesion assay

Primary cultures with keratinocytes from three different tonsilar tissues were established. The keratinocytes were cultured in keratinocyte growth medium (Cambrex, Walkersville, MD, USA) as previously described14. The third passaged cells were placed in human collagen type IV (Sigma-Aldrich, St. Louis, MO, USA) coated dishes and incubated for 10 min as previously described10. Rapidly adhering (R.A.) cells9, 15 were collected after vigorous washing and considered to be KSC fractions. After 90 min, suspended (non adhering, N.A.)9 cells were collected and considered to be TAC fractions that were post-mitotic differentiated cells10.

Annealing control primer (ACP)-based polymerase chain reaction (PCR)

Total RNAs extracted from the R.A. and N.A. samples were reverse transcribed for 1.5 h at 42℃ in a final reaction volume of 20µl containing 3µg of the purified total RNA, 4µl of reaction buffer (Promega, Madison, WI, USA), 5µl of dNTPs (each 2 mM), 2µl of 10µM dT-ACP1 primer (5'-CTGTGAATGCTGCGACTACGATIIIIIT(18)-3'), 0.5µl of RNasin (RNase Inhibitor (40 U/µl; Promega), and 1µl of murine leukemia virus reverse transcriptase (200 U/µl; Promega). The DEGs were screened by the ACP-based PCR method12, 13 using GeneFishing™ DEG kits (Seegene, Seoul, Korea). The PCR protocol for second-strand synthesis was one cycle at 94℃ for 1 min, followed by 50℃ for 3 min, and 72℃ for 1 min. The second-stage PCR amplification protocol was 40 cycles of 94℃ for 40 s, followed by 65℃ for 40 s, 72℃ for 40 s, followed by a 5 min final extension at 72℃. The amplified PCR products were separated on a 2% agarose gel stained with ethidium bromide. The differentially expressed bands were extracted and cloned into a TOPO TA cloning vector (Invitrogen, Carlsbad, CA, USA) and complete sequences were analyzed by searching for similarities using the BLASTX search program at the GenBank of the National Center for Biotechnology Information.

Western blotting

Total cell lysates (20 ug protein) form R.A. and N.A. cells underwent 8~16% SDS-PAGE and were transferred onto a PVDF membrane. To compare the differences in the expression of R.A and N.A. cells, other cells that had intermediate adherence ability were removed.

The DEGs, by ACP-based PCR, showed primarily mitochrondrial-related genes. Therefore, we investigated representative proteins related to mitochondrial biogenesis and to the respiratory chain including: mitochondrial complexes I to V, and peroxiredoxins 1 to 6. The primary antibodies included antibodies against mitochondrial complex I (NADH ubiquinone oxicoreductase 39 kDa a subcomplex 9, NDUFA9), complex II (Succinate-ubiquinone oxicoreductase 70 kDa flavoprotein, SDHA), complex III (Ubiquinone-cytochrome c, core II, UQCRC2), complex IV (cytochrome C oxidase subunit I, COXI and, subunit IV, COXIV) and complex V (ATP synthase, F1 complex a, ATP5A1) (all Invitrogen). In addition, antibodies against peroxiredoxins 1~6 (Prx 1~6) (Lab Frontier, Seoul, Korea), S-100 calcium binding protein A8 (Santa Cruz Bio, Santa Cruz, CA, USA) and β-actin (Sigma-Aldrich) were evaluated against the loading of proteins. The membrane was incubated with goat anti-mouse IgG or goat anti-rabbit IgG conjugated with horseradish peroxidase (Santa Cruz Bio). The immunoblots were developed by the ECL reaction (Amersham, Piscataway, NJ, USA).

Tetramethyl-rhodamine ethyl ester (TMRE) and mitotracker staining

R.A. and N.A. cells were stained with 150 nM TMRE (Invitrogen) in the dark at 37℃ for 30 min. The cells were then washed, resuspended in phosphophate-buffered saline containing 15 nM TMRE, and analyzed by flow cytometry (FACS Calibur, BD Biosciences, Franklin lakes, NJ, USA). For the mitotracker staining, the cells were cultured in chamber slides overnight, then washed and fixed with 3.7% paraformaldehyde (Sigma-Aldrich). The cells were stained with 500 nM of MitoTracker Orange CMTMRos (Invitrogen) in the dark at room temperature for 20 min after permeabilization with triton X-100 (Sigma-Aldrich). The fluorescent signal was visualized by a laser-scanning confocal microscope (LSM510, Germany).

ACP-based PCR for rapid adhering (R.A.) and non adhering (N.A.) cells

The products separated for the DEGs on agarose gels showed 10 different cDNA bands (C1-C10) by hatched blastocyst-specific expression (Table 1). The 10 different DEG clone found by comparing the bands of the R.A. and N.A. cells included four mitochondrion-related genes: C1 (mitochondrion), C2 (mitochondrion), C5 (mitochondrion) and C8 (mitochondrial DNA). In addition, C7 (S100 calcium binding protein A8) and C9 (nitric oxide synthase interacting protein) also differed in the DEG clones. However, C6 was not significantly different by sequence comparison.

Table 1
The products of 10 differential cDNA bands (C1~C10) by hatched blastocyst-specific expression

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Western blotting

Western blot analysis showed that S100 calcium binding protein A8 showed no difference in the protein expression when R.A. and N.A. cells were compared. Western blot analysis using antibodies associated with mitochondrial biogenesis showed that in all the samples with R.A. cells, the nuclear DNA (nDNA) encoded mitochondrial respiratory complex subunits; NDUFA9 of complex I and COX IV of complex IV were reduced compared to the N.A. cells (Fig. 1A). The Mitochondrial DNA (mtDNA)-encoded COX I subunit of complex IV was reduced or equivocal. Interestingly other nDNA-encoded subunits of complex II (SDHA), III (UQCRC2), and V (ATP5A1) did not differ in their expression in repeated experiments. The Western blot analysis of peroxiredoxin isoforms showed that Prx 1, Prx 2, and Prx 4 were consistently reduced compared to the N.A. cells; however, the Prx 3 and Prx 5 expression did not differ (Fig. 1B).

Fig. 1
Mitochondrial function in the R.A. and N.A. cells. Primary cultures of oral tonsilar mucosal keratinocytes were established and separated by their different adhesion to collagen type IV. The expression of mitochondrial proteins in R.A. and N.A. cells was confirmed by Western blot analysis. (A) The expression of mitochondrial complexes in R.A. and N.A. cells. (B) The expression of peroxiredoxin 1~6 in R.A. and N.A. cells.

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TMRE flow cytometry and confocal microscopy of mitochondria

The TMRE staining, representative of mitochondrial membrane potential showed that N.A. cells had stronger fluorescence by FACS (Fig. 2A). Mitotracker staining showed that the structure and content of mitochondria were not significantly different in the R.A. compared to the N.A. cells (Fig. 2B).

Fig. 2
TMRE flow cytometry and confocal microscopy for mitochondria. (A) R.A. (dotted line) and N.A. (solid line) cells were stained with TMRE and analyzed by FACS. The abscissa shows the intensity of the fluorescence, the ordinate the relative number of cells. (B) R.A. and N.A. cells were treated with MitoTracker Orange CMTMRos to stain the mitochondria and then studies under a confocal microscope.

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