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Department of Biochemistry and Molecular Biology, Johns Hopkins University, Baltimore, Maryland, USADepartment of Biological Chemistry, Johns Hopkins University, Baltimore, Maryland, USADepartment of Dermatology, School of Medicine, Johns Hopkins University, Baltimore, Maryland, USA
In 1978, Sun and Green carried out a densitometry-based analysis of PAGE samples and reported that keratin proteins account for 25–35% of total cellular proteins in human epidermal keratinocytes serially passaged in primary culture. This represents an astounding figure with very significant implications for the structural support role of keratin intermediate filaments in epidermis, and its regulation by posttranslational modifications and/or interaction with associated proteins. We report here on an effort to investigate this issue further, using a distinct method of keratin quantitation in sorted but otherwise native cell populations, and taking advantage of the significant progress made in our understanding of the structure of keratin filaments and differential regulation of keratin expression in epidermis.
Freshly isolated keratinocytes were obtained directly from 2-day old C57Bl/6 newborn mice, labeled with fluorochrome-conjugated antibodies to the surface marker integrin β-1, and then sorted by flow cytometry according to the expression level of this antigen (
), this integrin is primarily expressed in basal layer keratinocytes, and only poorly so in differentiating keratinocytes in the suprabasal layers of epidermis. We collected cells that express intermediate levels of integrin β-1 at their surface (Figure 1a). Such integrin β-1 “dim” cells (
) consist of basal cells with transit-amplifying proliferation status and represent the majority of basal layer keratinocytes. The less abundant cells that express surface integrin β-1 at a higher level (designated “bright”) are enriched in epidermal stem cells (
). Ultrastructural analysis shows that the sorted integrin β-1-dim cells are homogeneous, with a round shape and a smooth surface, and features a round and centrally located nucleus surrounded by thick bundles of keratin filaments in the cytoplasm (Figure 1b). The appearance of keratin filaments, in particular, is very reminiscent of those seen in basal layer keratinocytes in situ (
Figure 1Isolation and characterization of epidermal basal keratinocytes. (a) Keratinocytes harvested from C57Bl/6 P2 mouse skin were stained with fluorochrome-conjugated antibody to the surface protein integrin β-1 and sorted using flow cytometry. An integrin β-1-dim status reflects a transit-amplifying population of basal keratinocytes, whereas an integrin β-1-bright status denotes a subpopulation enriched in stem cells. (b) Transmission electron micrograph of a representative sorted basal (integrin β-1-dim) keratinocyte. Bar=1μm. (c) Immunofluorescence micrographs of P2 skin cross-sections highlight the occurrence of K17-positive basal cells in the hair follicle–proximal interfollicular epidermis (see arrows). Bar=10μm. (d) Western blots (WBs) show the presence of K17 but not K16 in FACS-sorted basal cells, given the protocol used (see a). “Std.” refers to relevant purified keratin standard. bc, basal cell; hf, hair follicle; Krt IFs, keratin intermediate filaments; N, nucleus; PE, phycoerythrin.
Sorted basal cells were lysed in urea lysis buffer and the resulting protein extracts were analyzed to measure the concentrations of K5, K14, and K15, the three major keratins in the basal progenitor cells of the epidermis. Serially diluted aliquots of native protein lysates were resolved by PAGE, and K5, K14, and K15 antigens were quantitated by infrared western blot analysis (Li-Cor Biosciences, Lincoln, NE). Calibration curves were established using purified recombinant forms of human K5, human K14, and mouse K15 as standards, taking advantage of the linear relationship between western signal intensity and protein concentration (Figure 2a–c). Relevant experimental details are given in Supplementary Materials online.
Figure 2Average keratin concentration and the number of keratin filaments in basal layer keratinocytes of epidermis. The keratin fraction of total cell proteins in sorted basal keratinocytes was determined using quantitative infrared western blot analysis. (a–c) Western blots show a linear relationship between keratin standard concentration and signal intensity for (a) K5, (b) K14, and (c) K15. (d) The keratin fraction of total cell proteins and the number of keratin monomers in sorted basal cells were calculated from these calibration curves. Data are presented as average±SD from eight (for K5) or six (for K14, K15) independent experiments. (e) Estimation of keratin amount/concentration and number of keratin filaments in epidermal basal keratinocytes. Additional data are given in Supplementary Tables S1–3 online. IFs, intermediate filaments.
The quantitative data obtained are related in part in Figure 2d and e and otherwise in toto in Supplementary Tables S1–3 online. We estimate that the average sorted basal keratinocyte contains 36.81±4.34pg of total protein, of which 4.91±1.07, 2.18±0.80, and 0.98±0.01pg, respectively, represent K5, K14, and K15 (Supplementary Table S3 online). This yields a 1.27:1 molar ratio for type II vs. type I keratin proteins, rather than the expected 1:1 ratio (
). This difference could be because of the presence of K17 (but neither K6 nor K16) in a small subset of sorted basal keratinocytes originating from the hair follicle–proximal interfollicular epidermis and the upper infundibulum (
; Figure 1c and d). Alternatively, it is formally possible that the antibodies we used have different affinities for mouse and human keratins (if so, the difference would be expected to be small; Supplementary Materials online), or that there is a small imbalance in the keratin I/II molar ratio when considering the total pool, instead of the polymerized pool, of keratins (see
We selected the data collected for K5 as a reference, and assumed a 1:1 molar ratio per keratinocyte for further calculations. Assuming 700 keratin monomers per micrometer length of filament (
; Supplementary Table S1 online), we estimate that the average basal keratinocyte contains 33,179±7,214 keratin filaments (Figure 2e). This estimate obviously depends on assumptions made about the length of individual filaments (Figure 2e). Even when factoring modest errors made in either the assumptions or the data obtained, it appears that there are tens of thousands of keratin filaments in basal keratinocytes of epidermis.
In our hands, keratins account for 17–27% of total cellular proteins (Figure 2d and e) in newborn mouse keratinocytes (i.e., up to 13.4%, 5.9%, and 2.8% for K5, K14, and K15, respectively), a finding that is close to what was reported by
for human keratinocytes more than 30 years ago. Using estimates of cell size, cell volume (and cytoplasmic volume) derived from measurements made on transmission electron micrographs of sorted cells (Supplementary Table S2 online), we calculate that the total concentration of these three keratins in the average nonstem basal keratinocyte is ∼40μgμl−1 or 520μM (Supplementary Table S3 online). By comparison, the total actin concentration ranges between 25 and 200μM in various cell types (
). The latter figures convey that the concentration of keratin in basal keratinocytes approximates that of actin in muscle tissue. Further, our assumptions and measurements together yield a total protein concentration of ∼180μgμl−1 in sorted keratinocytes (Supplementary Table S3 online), a figure that is consistent with previous reported values for mammalian cells (50–400μgμl−1;
), the corresponding number of protein monomers (∼1.9 million, Figure 2e) and concentration (∼10μM, Supplementary Table S3 online) is large, relative to the pool of most other cellular proteins. This sizable soluble pool is presumably available to sustain the remodeling of keratin filaments under steady-state conditions, and/or to fulfill nonstructural roles in the cell.
These quantitative figures are essential to a deeper understanding of keratin organization and function, and their regulation, in epidermis and related surface epithelia.
ACKNOWLEDGMENTS
We thank Janet Folmer for assistance with electron microscopy and the members of the Coulombe laboratory for their advice and support. This work was supported by grant AR42047 from the National Institute of Arthritis, Musculoskeletal, and Skin Diseases (to PAC). XF was supported in part by grant T32CA009110 from the National Cancer Institute.
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