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J Biol Chem, Vol. 273, Issue 52, 35176-35184, December 25, 1998
From the Keratins (K), the cytoplasmic intermediate
filament (IF) proteins of epithelial cells, are encoded by a multigene
family and expressed in a tissue- and differentiation-specific manner.
In human skin, keratinocytes of the basal layer of epidermis and the
outer root sheath of hair follicles express K5 and K14 as their main
keratins. A small subpopulation of basal cells exhibiting stem-cell
like characteristics express, in addition, K19. At 40 kDa, this keratin
is the smallest IF protein due to an exceptionally short
carboxyl-terminal domain. We examined the assembly properties of K19
and contrasted them to K14 in vitro and in
vivo. Relative to K5-K14, we find that K5-K19 form less stable
tetramers that polymerize into shorter and narrower IFs in
vitro. When transiently co-expressed in cultured baby hamster
kidney cells, the K5 and K19 combination fails to form a filamentous
array, whereas the K5-K14 and K8-K19 ones readily do so. Transient
expression of K19 in the epithelial cell lines T51B-Ni and A431 results
in its integration into the endogenous keratin network with minimal if any perturbation. Collectively, these results indicate that K19 possesses assembly properties that are distinct from those of K14 and
suggest that it may impart unique properties to the basal cells
expressing it in skin epithelia.
Keratins (K)1 are
intermediate filament (IF) proteins encoded by a large multigene family
and expressed in epithelial tissues. The >30 known keratins (40-70
kDa) expressed in soft epithelia have been subdivided into type I
(acidic, K9-K20) and type II (basic, K1-K8) based on DNA sequence
homology and gene structure (1). Keratin filament assembly is a
multistep process that begins with the formation of a type I-type II
heterodimer (2), distinguishing them from most other IF proteins, which
form homodimers. As a result, an epithelial cell must coordinately
express at least one type I and type II keratin genes in order to
assemble an IF network in its cytoplasm. Many keratin genes are in fact
regulated in a pairwise, differentiation-specific manner, creating
patterns that have been well conserved among mammalian species (1, 3). In stratified epithelia, for instance, the type II gene K5 and the type
I genes K14 and K15 are expressed in the basal layer, whereas distinct
combinations of type I and type II keratin genes are expressed in the
differentiating suprabasal layers. A major function of keratin IFs in
stratified epithelia is to contribute to the physical strength that is
necessary to maintain their integrity in response to normal load of
mechanical stress. This is particularly obvious in the epidermis and
oral mucosa, as mutations affecting specific keratin proteins underlie
several inherited blistering disorders such as epidermolysis bullosa
simplex, epidermolytic hyperkeratosis, oral white sponge nevus, and
others (2, 4, 5).
One of the most intriguing keratin is K19. At 40 kDa, this type I
keratin is the smallest known IF protein (6, 7). The primary structure
of human, bovine, and mouse K19 is highly conserved and made unique by
virtue of an exceptionally short (13-residue long) tail domain at the
carboxyl terminus (8-11). Otherwise, the length of the central rod
domain (312 residues), a major determinant of IF polymerization (1, 2),
is highly conserved, whereas the size of the non-helical,
amino-terminal head domain (72 residues) is within the range observed
for type I keratins (8-10). The primary structure of K19 is better
related to the type I epidermal keratins than to K18, which
predominates in simple epithelia (10). Still, the K19 gene is expressed
in a variety of unrelated cell types in both simple and complex
epithelia, such that there is no obvious relationship between the
presence of K19 protein and the structure or function of an epithelial
cell (10, 12-15). Although this combination of an unusual primary
structure and heterogeneous distribution renders K19 particularly
interesting, the function that it may perform remains unclear.
In adult human skin epithelia, K19 is found in a relatively small
subset of keratinocytes in the basal layer and in Merkel cells, a minor
population (<1%) of mechanosensory cells (Refs. 16 and 17 and
references therein). These two cell types are functionally distinct and
can be discriminated based on the specific reactivity of Merkel cells
with antibodies directed against K18 and K20 (16, 18, 19). In human
trunk skin, the K19-containing keratinocytes were found to localize to
a distinct region of hair follicles known as the bulge, where they are
confined to the basal layer of the outer root sheath. In the thicker
glabrous epidermis of palms and soles, K19-containing keratinocytes are
restricted to the deep portion of the rete ridges and are again
confined to the basal layer (20). Such a distribution is intriguing, as
both the bulge area of hair follicles and the deep portion of the
epidermis in glabrous skin were shown to contain the reservoir of stem
cells in adult skin (21, 22). Michel et al. (20) reported
that many of the [3H]thymidine label-retaining cells in
these areas of mouse skin actually express K19, raising the possibility
that a sizable fraction of the K19-expressing skin keratinocytes may be
stem cells. Here we examine the assembly properties of K19 at the
subunit, 10-nm filament, and cytoplasmic organization levels. The
results of our studies suggest that K19 possesses distinct assembly
properties that are likely to impact on the organization of keratin
filaments in the progenitor cells of the skin.
Immunological Analyses--
Frozen sections of human skin
samples, cultured newborn foreskin keratinocytes (23), or cultured cell
lines were analyzed by indirect immunofluorescence as described (20).
The following primary antibodies were used: mouse monoclonal antibodies
against K19 (Ks19.1, American Research Products, Belmont, MA, and clone A53-B/A2 from Sigma); K14 (Sigma and LL001; a gift from I. Leigh, Imperial Cancer Research Fund, London, UK); K18 (Ks18.174, American Research Products); K7 (LP5K, a gift from I. Leigh, Imperial Cancer Research Fund, London, UK); vimentin (V9, Sigma or anti-RLV, a gift
from N. Marceau, Centre Hospitalier Universitaire de Qúebec, Quebec); K8 and K18 (L2A1; Ref. 24) and CAM5.2 coupled to fluorescein (Becton Dickinson, San Jose, CA); guinea pig and rabbit polyclonal antibodies against K5 (25); rabbit polyclonal antiserum anti-peptide directed against K14 (Ref. 26; a gift from N. Marceau, Quebec, Canada),
against all K6 isoforms (27), and against K18 (28); and a guinea pig
polyclonal antibody reacting against K8 and K18 (03-GP11, American
Research Products). The goat secondary antibodies used were as follows:
fluorescein-conjugated anti-guinea pig (Jackson ImmunoResearch, West
Grove, PA), anti-mouse (Cedarlane, Hornby, Ontario, Canada),
anti-rabbit (Chemicon, Temecula, CA), and rhodamine-conjugated anti-mouse (Chemicon). Negative controls consisted of omission of the
primary antibody during the labeling reaction. For immunoelectron microscopy of methanol-fixed transfected BHK cells, a 10-nm gold goat
anti-mouse antibody (British Biocell International, Cedarlane, Hornby,
Ontario, Canada) was used for detection, and samples were fixed with
2.5% glutaraldehyde for 15 min, washed, post-fixed with 2%
OsO4 for 30 min, embedded in Epon and observed with a JEOL
1200 EX operated at 80 kV. Western blots were revealed by alkaline
phosphatase-conjugated secondary antibodies (Sigma).
Cloning of Human K19 cDNA--
The entire coding sequence
for human K19 was obtained by reverse transcriptase-PCR using
oligonucleotide primers designed from the published sequences (9, 10)
and total RNA isolated from MCF-7 cells as described (29). The PCR
product obtained was subcloned in plasmid pET-3d (30) for expression in
bacteria. Several clones were subjected to dideoxy sequencing. The
cDNA clone selected (see "Results") was transferred into
GW1-CMV (31) for expression in cultured mammalian cells.
Keratin Expression and Purification--
We used an
Escherichia coli expression system based on the phage T7 RNA
polymerase gene (30) to produce recombinant human keratins from
plasmids pET-K19 (this study), pET-K5, and pET-K14 (32). Recombinant
keratins were purified to near-homogeneity as described (31-33).
Heterotypic Complex Formation and Analysis--
Purified
recombinant type I and type II keratins were mixed in a ~45:55 molar
ratio at In Vitro Keratin Filament Assembly, Negative Staining, and
Electron Microscopy--
Mono Q fractions containing heterotypic
keratin complexes were used for in vitro polymerization
assays (32). Polymerization was achieved by dialysis of samples at 300 µg·ml Transient Expression of Keratin cDNAs in Cultured Cells in
Vitro--
Keratin cDNAs were inserted into the GW1-CMV expression
plasmid (31). pMK8 and pMK18 (kindly provided by B. Omary) are
derivatives of the CMV promoter-based pMRB101 expression plasmid (34).
For transient transfection assays, all cell lines were cultivated in
media supplemented with 10% fetal bovine serum and antibiotics unless
otherwise indicated. BHK-21 cells and NIH3T3 mouse fibroblasts were
grown in Dulbecco's modified Eagle's medium, PtK2 (35), and A431
cells in Dulbecco's modified Eagle's medium:Ham's F12 (3:1) medium
and T51B cells, an established cell line derived from adult rat liver
as well as T51B-Ni, a subclone selected by prolonged exposure of the
parental cell line to nickel subsulfide (Ref. 36; a gift from N. Marceau, Quebec, Canada) were cultivated in Quantitation of Epidermal Keratins in Transfected Cells--
For
quantitation of transfected keratins, BHK, PtK2, and A431 cells were
seeded on 100-mm plates that contained one 22-mm2 glass
coverslip. BHK cells were cotransfected with the following CMV plasmid
combinations: K5 and K14, K5 and K19, K14 and K19, or CMV vector alone.
PtK2 and A431 cells were transfected with each of control CMV, CMV-K14
cDNA, and CMV-K19 cDNA plasmids. The amounts of DNA and the
volume of precipitates were scaled up on a per surface area basis to
maintain conditions similar to those prevailing in 35-mm culture
dishes. At 26-30 h post-transfection, the glass coverslips were
processed for staining to determine transfection efficiency. The
remaining cells were recovered by scraping directly in gel loading
buffer (total protein extracts). For SDS-PAGE/immunoblot analyses,
known amounts of purified recombinant K14 or K19 (10, 20, 40, and 60 ng) were co-electrophoresed along with extracts prepared from
transfected cells and blotted onto nitrocellulose. Blots were
double-stained for the endogenous K8-18 (PtK2 cells) or vimentin (BHK
cells) to ensure that equal amount of proteins were loaded in each
lane. At each step, blots were scanned and densitometric analyses done
using NIH image.
K19 Is Co-expressed with K5 and K14 in a Subset of Keratinocytes in
Situ and in Vitro--
Double immunofluorescence microscopy was
performed to identify the type II keratins that occur in the subset of
K19-positive basal keratinocytes. Our studies focused on the type II
keratins K5, K6, K7 and K8, as these are known to occur in glabrous and hairy skin (3, 12, 38, 39). We did not attempt to localize either K1
and K2e, as these are restricted to the suprabasal differentiating layers in epidermis and in the uppermost segment of the hair follicle outer root sheath (see Refs. 40 and 41 and references therein). As
expected (data not shown), we found that K19 co-localizes with K5 and
K14 in the outermost (basal) cells of the bulge region of hair
follicles (10, 42) and in the basal layer of newborn foreskin
epidermis. We did not find evidence of co-expression of K19 with either
K6 or K7 (data not shown), whereas a very scarce signal for K8 was
detected in the hair bulge area, consistent with the relative paucity
of Merkel cells in this region (16). In primary cultures of human
foreskin keratinocytes, we find that K19 is expressed in a subset of
cells (20) and co-localizes with K5, K6, and K14 (not shown). Under our
culture conditions, only a proportion of K19-containing cells also
express K8, and we do not detect immunoreactivity for K7 (data not
shown). From these studies, we conclude that K5 is likely to be the
major type II keratin partner for pairing and co-assembly with K19 in
the relevant subset of human skin keratinocytes and that the type I K14
is also present in this cell population.
Characterization of the Human K19 cDNA Clone and Its Protein
Product--
We applied reverse transcriptase-PCR on total RNA from
human MCF-7 breast adenocarcinoma cells to clone the coding sequence of
K19. We identified a subclone whose nucleotide sequence is in complete
agreement with that previously published by Eckert (9) with one
exception. The PCR-mediated creation of a NcoI restriction
site (CCATGG) at the start codon resulted in a Thr (ACT)
Comparing K5-K19 and K5-K14 Heterotypic Complex Formation in
Vitro--
To compare the formation of heterotypic complexes involving
K5-K19 and K5-K14, these purified keratins were mixed in a
predetermined ratio and subjected to established chromatography and
chemical cross-linking assays (32, 33). When subjected to Mono Q
chromatography at pH 8.1 in the presence of 6.5 M urea,
purified type II epidermal keratins exist as monomers and elute at
~70-80 mM Gdn-HCl, designated as "peak 1," whereas
purified type I epidermal keratins (also monomeric) elute at a higher
salt concentration (~100-130 mM Gdn-HCl), designated as
"peak 2." The sequential elution of type II and type I keratins
under basic buffer conditions is consistent with their known surface
charge (12). Mixtures of type I and type II keratins show a different
elution profile as follows: heterotetramers elute at a yet higher salt
concentration (~150-175 mM Gdn-HCl), designated as "peak 3", whereas heterodimers co-elute with type I
monomers in peak 2 fractions (Ref. 33 and references therein).
When tested individually in the Mono Q chromatography assay, K19 elutes
at the position characteristic for monomeric type I keratins (peak 2;
data not shown). As expected, no dimer or tetramer is seen when
purified K19 is subjected to cross-linking (data not shown). When mixed
with K5 prior to chromatography, on the other hand, K19 shows an
elution profile consistent with its recruitment in heterotypic
complexes. Indeed, the majority of the K5 and K19 proteins elute as a
1:1 complex at a higher Gdn-HCl than either keratin alone, indicating
efficient heterotypic complex formation (Fig.
2A). Consistent with the
slightly less acidic character of K19 monomer compared with K14 (data
not shown), the bulk of K5-K19 heterotypic complexes elute at
Chemical cross-linking using BS3 establishes that the major K5-K19
species formed under 6 M urea buffer conditions is a
We next examined tetramer formation under conditions where K19 and K14
would have to compete for a limiting amount of K5. Purified K19, K14,
and K5 were mixed in a ~1:1:1 ratio in the presence of 6.5 M urea, incubated for 1 h, and subjected to Mono Q
chromatography. Consistent with the high efficiency with which K19 and
K14 each pair with K5, the latter is almost entirely recruited (94%)
to peak 3 fractions ("tetramer") (Fig.
3). The K5-containing heterotypic
complexes elute as a single peak, suggesting the formation of mixed
heterotetramers. Densitometry analysis of all the fractions making up
peak 3 revealed the presence of a greater amount of K14 on a molar
basis, such that it is enriched 1.7-fold compared with K19. As
expected, this ratio is inverted in favor of K19 in the peak 2 fractions, which contain the molar excess of type I keratins (Fig. 3).
These data suggest that K5 has a greater affinity for K14 compared with
K19, a finding that may have implications for the regulation of K14 and
K19 in skin epithelia in vivo.
Comparing K5-K19 and K5-K14 Filament Assembly Properties in
Vitro--
To assess filament formation in vitro, purified
type I-type II heterotypic complexes were subjected to dialysis against
standard epidermal assembly buffer and examined by negative staining
and electron microscopy. The K5-K19 pair readily assembles into
intermediate-sized filaments (Fig. 4),
although less efficiently (75%) than the K5-K14 pair (>90%) as
shown by a pelleting assay (data not shown). The K5-K19 filaments are
significantly narrower than the K5-K14 ones (8.4 ± 0.1 nm
versus 9.9 ± 0.1 nm, respectively; mean ± S.E.). Although it is consistent with the mass difference between K19 (40 kDa)
and K14 (50 kDa), this smaller diameter could also reflect a smaller
number of subunits per cross-section, a possibility that we did not
investigate further. The average length of K5-K19 filaments (1.1 ± 0.1 µm) is shorter than that of K5-K14 filaments (2.0 ± 0.1 µm), a statistically significant difference (p < 0.01). It is likely that the sampling for these measurements is biased toward short filaments. Still, the numbers obtained reflect a trend
that is readily detectable from a qualitative inspection of K5-K14 and
K5-K19 assemblies (Fig. 4).
As predicted from our theoretical understanding of the polymerization
of fibrous polymers (43), shorter filaments may arise following
mechanisms as distinct as increased nucleation, decreased rate of
elongation, or decreased stability of the polymer at steady state (44).
We did not address these various possibilities at an experimental
level. However, the lower efficiency of K5-K19 assembly compared with
K5-K14 (as measured under assembly conditions optimal for epidermal
keratins (45, 46) implies the existence of a higher concentration of
unpolymerized proteins at steady state and supports the idea that
K5-K19 filaments are less stable than K5-K14 ones. We examined
assembly under increased ionic strength buffer conditions (50 mM Tris-HCl), which are optimal for the in vitro
polymerization of wild-type K8-K18 (47) and truncated forms of K5-K14
proteins (48). Under such conditions, K5-K14 typically form ball-like
aggregates of filaments, whereas K5-K19 form a mixture of looser
aggregates and bundles in which multiple filaments are intertwined
around one another (data not shown). We conclude from these studies
that K5-K19 can readily polymerize under standard epidermal keratin
assembly buffer conditions in vitro but that the resulting
filaments are shorter than K5-K14 ones, perhaps as a result of a
lesser stability.
Assembly Properties of K19 in Non-epithelial Cells in
Culture--
We compared the assembly properties of various keratin
combinations involving K5 and K8 as type II keratins and K19, K14, and
K18 as type I keratins in transfected BHK-21 cells. This cell line has
a type III (vimentin and desmin) but lacks a keratin IF network (49),
enabling us to examine the ability of combinations of type I-type II
keratin to polymerize de novo in the cytoplasm of a living
cell. Upon co-transfection, the majority of cells (>90%)
co-expressing K5 and K19 do not show a filamentous array but a
combination of dots distributed throughout the cytoplasm and aggregates
preferentially located around the nuclei (Fig. 5, A and A'). In
contrast and as previously reported (31), a majority of transfected
cells (~70%) co-expressing K5 and K14 show bundles of keratin
filaments extending throughout the cytoplasm (Fig. 5, B, B',
and C). We examined BHK cells at times ranging from 24 to
48 h after transfection and did not observe significant differences in the proportion of cells showing aggregated
versus filamentous organization. We could not detect any
obvious difference in the organization of the vimentin network in
K5-K19- and K5-K14-transfected cells (data not shown). Expression of
the K5-K19 and K5-K14 plasmid combinations in NIH 3T3 cells produce a
very similar outcome (data not shown).
We compared the K19 and the K14 protein levels in BHK cells transfected
with the K5-K19 or K5-K14 plasmid combinations (see "Materials and
Methods") and found that they were comparable (Fig. 6A). Moreover, transfection of
various molar ratios of K5:K19 plasmids (up to 5:1) did not improve
filament forming potential in BHK cells. These data suggest that it is
unlikely that the K5-K19 aggregates are produced as the result of K19
overexpression. Electron microscopy of double-transfected BHK cells
shows that the large aggregates that are immunopositive for K19 are
made of non-filamentous proteins (Fig. 6B). Collectively
these data indicate that under our transfection conditions, and in
contrast to the K5-K14, K5-K16, and K6-K14 plasmid combinations
(this study and Ref. 31), co-expression of K5 and K19 does not give
rise to a filamentous array in cultured BHK-21 cells.
We next examined whether K8, the major type II keratin gene
co-expressed with K19 in simple epithelia, is a "better" pairing partner for the latter in transfected BHK cells. We found that in
contrast to K5-K19, only ~12% of cells co-transfected with K8-K19
show aggregates (not shown). Among the other cells, ~33% show a
dispersed filamentous array in a fashion analogous to K5-K14, whereas
~55% show thick bundles of filaments (Fig. 5, D and
D'). By comparison, we found that ~60% of BHK cells
co-transfected with K8 and K18 plasmids show a well extended network of
filaments (Fig. 5E), whereas ~20% show large bundles of
filaments (data not shown). Finally, 30% of the BHK cells transfected
with K8 and K14 plasmids featured a filament network (Fig.
5F). These results establish that as is the case in
vitro (47, 50-52), the K19 cDNA clone gives rise to a
polymerization-competent keratin when paired with K8 in an in
vivo like setting.
Transient Expression of K19 in Selected Epithelial Cell Lines Leads
to Its Integration into the Pre-existing Keratin Network--
Since
the K5-K19 pair seemed unable to form filaments de novo in
fibroblastic cells, we tested the ability of K19 to integrate into the
pre-existing keratin network of an epithelial cell. We transfected the
K19 cDNA into the T51B and T51B-Ni epithelial cell lines derived
from rat liver and which express K8, K14, and vimentin but are negative
for K19. In addition, T51B-Ni cells express K18 and features a more
extended keratin network (36). In either cell line, transient
expression of the K19 cDNA leads to integration of K19 protein into
the endogenous network in >70% of the transfected cells (Fig.
7, A and A';
T51B-Ni). Similarly, transfection of the K18 cDNA results in a K18
protein-containing filamentous array in >80% of transfected cells
(Fig. 7, B and B'; T51B-Ni).
We next examined the behavior of transfected K19 in the A431 human
epidermoid carcinoma cell line, which expresses K5, K6, K7, and K8 as
their main type II keratins (12). Under our culture conditions, A431
cells express K18 at high levels, are negative for K19, and show
sporadic expression of K14 (only ~10% of cells). Transient
expression of the K19 (Fig. 7, C and C') and K14 (Fig. 7,
D and D') cDNAs results in a filamentous
pattern for 80 and 95% of transfected A431 cells, respectively,
without disruption of the endogenous K8-18 network. Quantitation of
transfected keratins by Western blot analysis demonstrates that K19
protein is expressed to the same level as the transfected K14 in this
cell line (not shown). These studies establish that as expected of a
wild-type keratin, newly synthesized K19 can incorporate into a well
extended keratin IF array in these epithelial cell lines without
perturbing it in an obvious fashion.
Transient Expression of K19 Causes a Collapse of the Keratin
Network in PtK2 Epithelial Cells--
We transfected K19 cDNA into
the well characterized kidney rat kangaroo PtK2 cell line, whose well
extended K8-K18 filament network makes it attractive for the
examination of keratin assembly properties (e.g. Refs. 31
and 53-55). PtK2 cells transfected with CMV-K19 display a very
striking phenotype. In more than 80% of transfected cells, indeed,
expression of K19 results in the appearance of aggregates (Fig.
8, A and B) that
stain positively with antibodies directed against endogenous K8-K18
(Fig. 8, A' and B'). In contrast, and as shown
previously (31), human K14 protein integrates into the pre-existing
K8-K18 network without disrupting it in 94% of transfected PtK2 cells
(Fig. 8, C and C').
The observations made in PtK2 cells are surprising since K8 is a
"natural" partner for K19 in vivo, and these two
keratins form an IF array when co-transfected in BHK cells (Fig. 5).
Again, we compared the levels of exogenous keratins in transfected PtK2 cells and found that on average, K19 is expressed 5-fold more than K14
(not shown). It may be, therefore, that the unexpected phenotype seen
in K19-expressing PtK2 cells is due to its expression at significantly
higher levels. In support of this possibility, co-transfection of K8
and K19 produces a filamentous array in 70% of transfected PtK2 cells
(Fig. 8D), and transfection of the K18 plasmid alone results
in the production of clumps that correlate with a relatively abundant
expression as judged from immunofluorescence staining (not shown). In
contrast to K8, however, cotransfection of K5 and K19 fails to improve
the filament-forming properties of the latter, since 75% of
transfected PtK2 cells show either dots or clumps (Fig. 8E).
Although our observations suggest that the PtK2 cell line can be
sensitive to imbalances between type I and type II keratin protein
levels, this phenomenon appears to be keratin-specific. The results
obtained in this cell line support the notion that K14 and K19 behave
differently when co-expressed with K5.
There is increasing experimental evidence supporting the notion
that although a general function of keratin IFs is to impart mechanical
resilience to cells and tissues, various keratins possess different
properties that are exploited in specific ways by the epithelial cells
expressing them. For instance, the keratin pairs K5-K14, K1-K10,
K6-K16, and K8-K18 have intrinsic properties that clearly distinguish
them in vitro and that relate to their organization in
vivo (31, 33, 46, 47, 55-59). In a significant step, direct
support for this notion has recently been obtained through keratin
replacement experiments performed in transgenic mice. Indeed, Hutton
et al. (60) showed that human K18 is not able to fully
rescue the severe phenotype associated with a null mutation in the K14
gene of mouse (61) under conditions where an exogenous epidermal
keratin protein can. Similar studies in progress show that although it
does so more efficiently than K18, human K16 cannot entirely rescue the
mouse K14 null phenotype
either.3 Future efforts along
these lines should firm up the evidence substantiating the existence of
a significant relationship between keratin protein expression, filament
network organization and regulation, and epithelial function.
Here we report that the human type I keratin 19, which is distinct by
virtue of an extremely short tail domain (see Introduction), has
properties that are distinct from those of K14 in vitro and in vivo. We believe that may potentially translate into a
distinct keratin filament organization in the subset of skin
keratinocytes that co-express K5, K14, and K19 in vivo.
Relative to K5-K14 and under standard epidermal keratin assembly
conditions in vitro, indeed, K5-K19 forms less stable
tetramers that polymerize into shorter and narrower filaments with a
20% lower efficiency. Whereas numerous mechanisms could account for
these differences, our in vitro findings suggest that a
slightly lower stability of the K5-K19 polymer is potentially one of
them (see "Results"). To our surprise, we also found that
co-expression of K5 and K19 in two non-epithelial cell lines (BHK;
NIH3T3) does not give rise to the formation of IFs as assessed by
electron microscopy. This finding is at variance with the outcome of K5
and K19 co-polymerization in vitro, the co-expression of K8
and K19 in the same non-epithelial cell hosts (this study; see Refs. 50
and 51), and the expression of K19 in A431 cells, an epidermal cell
line that features K5 and K8 as type II keratins. Of relevance, Hutton
et al. (60) reported that expression of human K18 within the
basal layer of transgenic mouse epidermis causes the formation of
K5-K18 containing protein aggregates in the absence but not in the
presence of endogenous K14. Again, this finding contrasts with the
assembly behavior of purified recombinant human K5 and K18 in
vitro, which produce long filaments and no aggregates (60).
Collectively, our studies suggest that the impact of K19 expression
upon the organization of keratin IFs is context-dependent,
in that it likely depends upon the complement of keratin proteins,
their abundance, or the complement of IF-associated and IF-regulating
proteins present in the host cell.
Prior to our study, the assembly properties of K19 had for the most
part been compared with those of K18, the major type I keratin with
which it is co-expressed in simple epithelia. These studies showed that
K19 is capable of forming typical IFs in vitro and in
vivo when paired with K8, the major type II keratin present in
those cells (47, 50-52). How the intrinsic properties of K19 compare
with those of K18 remains unclear, however. In an elegant in
vitro study, Hofmann and Franke (52) demonstrated that K18 and K19
are distinct in their affinity, polymerization kinetics, and properties
of the filaments formed when paired with K8. In particular, they found
that K8-K19 filaments display a significantly lower viscosity and
appear shorter when examined by electron microscopy (see Fig.
7A in Ref. 52). In earlier studies, Lu and Lane (50) had
found that when expressed via retroviral vectors in NIH3T3 cells,
K8-K18 were able to form a more extended array of filaments compared
with K8-K19. A different conclusion was reached by Bader et
al. (51), however, whose experiments involved stably transfected fibroblasts, and more recently by Magin et al. (62) who,
from their electron microscopy analysis of the uterine epithelium in K18 null mice, concluded that the morphological features of K8-K19 IFs
were indistinguishable from K8-K18. The absence of consensus among all
these studies, including ours, may simply reflect the notion that as
speculated above, the assembly properties of individual keratins are
modulated by the co-assembly partner(s) involved, the complement of
keratin-associated and keratin-regulating proteins, or other features
particular to each cell type. It is reasonable to conclude, then, that
as is potentially the case in skin keratinocytes, the presence of K19
in simple epithelial cells may under some conditions promote a distinct
organization of keratin IFs.
Recent work from Oshima and colleagues (63) and Omary and colleagues
(64) showed that K18 and K19 are substrates for caspases, which are
proteases involved in programmed cell death. Given that caspase-mediated cleavage occurs at the VEVD sequence motif located in
the linker sequence between subdomains 2A and 2B, the resulting truncated K19 would likely behave as a dominant negative mutant (2). We
did not see the nuclear changes characteristic of apoptosis in K5-K19
expressing fibroblasts, and the K5-K14 combination gave rise to
filament arrays in a majority of transfected fibroblasts despite the
occurrence of a highly related motif (VEMD) in linker L12 of K14
protein. Thus, we do not think that this mechanism accounts for our
findings in transfected fibroblasts. On the other hand, we believe that
the lack of an extended tail domain in K19 may likely contribute to
specify the intrinsic assembly properties revealed in ours and other
studies. This speculation is supported by experimental evidence
implicating the tail domain of type I keratins such as K14 and K18 as
playing a role in filament stability (47, 48, 50).
Our demonstration that K14 and K19 possess distinct properties leads to
the speculation that the latter may promote a distinct organization of
the keratin IF network in the relevant subset of basal skin
keratinocytes. Michel et al. (20) recently extended the
original findings of Stasiak et al. (10) in showing that a
fraction of the K19-expressing basal cells in mouse skin are thymidine
label-retaining cells, an operational criteria for stem cell character.
This K19-positive subpopulation of cells localizes to the deep portion
of rete ridges in the epidermis of glabrous human skin and the bulge
portion of hair follicles in hairy skin (10, 20, 65). Interestingly, a
subset of "K14-low," less differentiated basal cells in the
outermost "basal" layer of the hair follicle outer root sheath has
been described by one of us (66), and these cells have been postulated
to serve as progenitors for both the hair follicle outer root sheath
and the epidermis. Electron microscopy studies in progress in one of
our laboratories (L. G.) confirmed the existence of these peculiar
basal cells in the bulge region of human hair follicles (data not
shown). Of note, there is no evidence for large keratin aggregates in keratinocytes localized in regions associated with K19 expression (66).4 Taken together, these
observations raise the possibility that in addition to their ability to
retain thymidine label, the subset of K19-expressing basal
keratinocytes may be less differentiated, express K14 at lower levels,
and show a distinct organization of keratin IFs. Further studies will
be required to address this intriguing possibility.
Expression of K19 occurs in a number of epithelial settings and
frequently correlates with a phenomenon of "plasticity" in epithelial cell fate or function (10, 65). In contrast to many of the
keratin genes expressed in the epidermis (2, 4, 5), mutations that
affect the primary sequence and function of K19 have not yet been
discovered in the human population. Moreover, introduction of a null
mutation in the K19 gene of mouse has no obvious consequence for the
morphogenesis and homeostasis of skin and other epithelia, at least
under basal conditions.5 The
function of K19 thus remains to be defined. Further characterization of
the morphology, properties, and function of the relevant subpopulation of skin epithelial cells in K19 null mice may provide clues for the
function of K19 in this tissue.
We are very grateful to Drs. Normand Marceau
for providing cell lines and antibodies; M. Bishr Omary for providing
antibodies and cDNAs; Irene Leigh and Elaine Fuchs for providing
antibodies; Claude Marin and Aristide Pusterla for photographic and
electron microscopy assistance, respectively; and Dr. Olivier Bousquet for helpful discussions. We also thank Drs. Gabriel Gosselin, Jean-Guy
Laberge, Jean-Marc Laliberté, and Alphonse Roy for providing skin specimens.
*
This work was supported by National Science Foundation Grant
MCB-9319560 and National Institutes of Health Grant AR44232 (to P. A. C.) and by Medical Research Council of Canada Grant MT-12087 (to L. G.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Recipient of a Studentship from the Medical Research Council of Canada.
¶
Recipient of a Scolarship from the Fonds de la Recherche en
Santé du Québec. To whom reprint requests should be
addressed: Laboratoire d'Organogénèse Expérimentale,
Hôpital du Saint-Sacrement, 1050, Chemin Sainte-Foy,
Québec, Canada, G1S 4L8. Tel.: 418-682-7696; Fax: 418-682-8000;
E-mail: lucie.germain{at}chg.ulaval.ca.
**
Recipient of a Junior Faculty Research Award from the American
Cancer Society.
The abbreviations used are:
K, keratin; IF, intermediate filament; Gdn-HCl, guanidine HCl; PAGE, polyacrylamide gel
electrophoresis; 2
Available on-line at the following address:
http://rsb.info.nih.gov/nih-image.
3
R. Paladini and P. A. Coulombe, unpublished data.
4
J. Fradette and L. Germain, unpublished data.
5
Drs. M. Taketo and Y. Tamai, personal communication.
The Type I Keratin 19 Possesses Distinct and
Context-dependent Assembly Properties*
§,¶,
, and**
Laboratoire de Recherche des Grands
Brûlés/LOEX, Hôpital du Saint-Sacrement,
Québec G1S 4L8, the Department of Surgery, Université
Laval, Sainte-Foy, Québec, Canada, and the Departments of
Biological Chemistry and Dermatology, The Johns Hopkins University
School of Medicine, Baltimore, Maryland 21205
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
250 µg·ml
1, incubated for 1 h at
room temperature, and fractionated by anion-exchange chromatography on
a Pharmacia Mono Q column (31-33). Fractions containing type I-type II
heterotypic complexes were pooled, dialyzed, and subjected to chemical
cross-linking using BS3 (bis-(sulfosuccinimidyl) suberate; Pierce) as
described (31, 33). Under these conditions, individual keratins do not
cross-link into larger size products. Cross-linked products were
resolved on a 4-16% gradient SDS-PAGE and stained with Coomassie
Blue. Densitometry was performed using the public domain NIH Image 1.59 software2 to estimate the
relative yield of each major product of the reaction. For the
competition assays, 300-435 µg of K14, K19, and K5 were mixed in
a molar ratio of 1:1:1 to generate a final mixture having a 280 µg·ml1 protein concentration. The mixture was
analyzed by chromatography and SDS-PAGE as described above.
Calculations and densitometric analyses were performed using NIH Image.
1 against 9 M urea, 50 mM Tris-HCl, 5 mM 
-ME, pH 7.6, at room temperature and then against 3 mM Tris-HCl, 10 mM -ME, pH 7.2, at 4 °C, for 16-24 h. In some
experiments, the ionic strength of the final buffer was 50 mM Tris-HCl. Polymerized keratins were visualized by
negative staining (1% uranyl acetate) on a Zeiss EM10A electron
microscope (31). For filament length determination, negatives
(magnification, × 20,000) were scanned and analyzed using the NIH
image software. Measurements were made on a random sample of 74-117
filaments from two independent experiments. For filament width
determination, 50 filaments were randomly selected from photographic
prints (magnification, × 61,250) collated from three independent
experiments. Statistical analyses were done using Student's
t test. Polymerization efficiency was determined via a
pelleting assay on the final assemblies as described (31, 33).
- minimum Eagle's
medium supplemented with 5% fetal bovine serum (Immunocorp.).
Transfections were done on subconfluent cells grown on
22-mm2 glass coverslips using the calcium phosphate
precipitation method (31) or a polycation-mediated gene transfer using
polyethylenenimine (Aldrich, Milwaukee, WI) (37). At 24-72 h
post-transfection, cells were fixed with absolute methanol for 20 min
at 
20 °C and processed for indirect double immunofluorescence as
described above. As routine controls, CMV-GW1 or mock-transfected cells were processed in parallel with the relevant antibodies. For the epithelial cell lines that already express endogenous keratins, we
cotransfected vectors containing keratins cDNAs with limiting amount (1:9 ratio GFP:total DNA) of pEGFP-N1 vector
(CLONTECH, Palo Alto, CA) and relied on the
intrinsic fluorescence of the green fluorescence protein to identify
transfected cells after fixation with 1% formaldehyde for 10 min and
cold methanol for 20 min.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Ala (GCC) substitution at the first amino acid. When induced, BL21
(DE3) E. coli transformed with plasmid pET-K19 (see
"Materials and Methods") express a 40-kDa protein that is
retrieved in the inclusion body fraction and which co-purifies with
acidic keratins on a Mono Q anion exchange column (31-33). Purified
K19 reacts with a monoclonal antibody to K19 by Western blotting, and
the positive recombinant antigen co-migrates with native K19 extracted from cultured human MCF-7 cells (Fig. 1).
These data establish the validity of our cDNA clone as well as of
the recombinant protein derived from it.
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Fig. 1.
Immunological analysis of purified human
recombinant K19. Western blot analysis using a monoclonal antibody
reacting against human K19. A, human recombinant K19 (50 ng). B, MCF-7 cell extract (~1.5 µg of total proteins).
The purified recombinant protein co-migrates with endogenous (native)
K19 from total extracts of MCF-7 cells.
145-150 mM Gdn-HCl, whereas the peak of K5-K14 complex
occurs at 155-160 mM Gdn-HCl (Fig. 2A). In
addition, we find that relative to K5-K14, a small fraction of the K5
and K19 proteins co-elute significantly earlier in the Gdn-HCl
gradient, in the so-called peak 2 fractions (Fig. 2A). This
suggests that K5-K19 may not form heterotetramers with the same
efficiency as K5-K14.
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Fig. 2.
A, elution profiles of recombinant
keratins during anion-exchange chromatography. Purified K5 and K19
(top) and K5 and K14 (bottom) were mixed in a
55:45 ratio (see "Materials and Methods"), applied to a Mono Q
anion-exchange chromatography column, and eluted over a 0-200
mM linear gradient of Gdn-HCl. Aliquots were analyzed by
SDS-PAGE followed by staining with Coomassie Blue. Peak 1,
excess type II keratins; peak 2, type I keratins and/or
heterodimers of type I-II keratins; peak 3, heterotetramers
of type I-II keratins. As shown previously (32), K5-K14 forms
heterotypic complexes with high efficiency and which elutes as a single
peak (peak 3). The K5-K19 mixture also readily forms
heterotypic complexes, of which a small fraction elutes at a lower
Gdn-HCl concentration (peak 2). B, chemical
cross-linking of type I-type II keratin heterotypic complexes. Type
I-type II heterotypic complexes isolated by anion-exchange
chromatography were dialyzed against 25 mM sodium phosphate
buffer containing either 6 or 8 M urea at pH 7.4. The
cross-linking agent BS3 was added to keratin complexes (200 µg·ml 1) at a final concentration of 5 mM
and the reaction stopped after 1 h. Cross-linked products (4 µg
of proteins) were resolved on a 4-16% gradient SDS-PAGE and stained
with Coomassie Blue. T, heterotetramers (~240 kDa);
D, heterodimers (~135 kDa); and M, monomers.
Although the K5-K19 combination forms tetramers with high efficiency
under 6 M urea buffer conditions, these are more readily
destabilized than the K5-K14 ones when the concentration of urea is
raised to 8 M.
240-kDa product, with a lesser amount of a 130-kDa product (Fig.
2B). As previously shown (e.g. Ref. 33), these
species correspond to covalently cross-linked heterotetramers (46%)
and heterodimers (38%) of keratins, respectively. Comparatively, the
K5-K14 pair forms the 240-kDa heterotetramers with higher
efficiency (70%). Furthermore, tetramers comprised of K5-K19 are
destabilized to a greater extent when the urea concentration is raised
to 8 M under otherwise identical conditions. Indeed, the
yields of tetramer cross-links for K5-K19 and K5-K14 are lowered to 5 and 48%, respectively (Fig. 2B). Since the rod domains of
human K19 and K14 feature comparable numbers of Lys residues (13 versus 18, respectively), it is unlikely that the difference
seen in their behavior is due to a bias arising from the mechanism of
cross-linking. This interpretation is further supported by the elution
profile of K5-K19 and K5-K14 from the Mono Q column (Fig.
2A). Taken together, these data suggest that K19 can readily
form heterotetramers in combination with K5 but that these are slightly
less stable than those formed by K5-K14.
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Fig. 3.
Assessing K19 tetramer-forming properties in
a competitive environment in vitro. For the
competition assay, 300-435 µg of purified recombinant K14, K19,
and K5 were mixed in a molar ratio of 1:1:1 to generate a final
mixture having a 280 µg·ml1 protein concentration.
After incubation at room temperature for 1 h, the mixture was
subjected to anion-exchange chromatography and eluted over a 0-200
mM linear gradient of Gdn-HCl. Fractions were analyzed by
SDS-PAGE and Coomassie Blue staining. Peak 1 represents the
elution position of type II keratins; peak 2, the excess of
type I keratins; and peak 3, the heterotypic complexes
containing the three keratins. Determination of the protein ratio in
the starting mix (SM) and in peak 3 fractions
shows that compared with K14, K19 has a 1.7-fold lower affinity for
K5.
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Fig. 4.
Electron microscopy of in vitro
reconstituted keratin filaments. Purified heterotypic
complexes of K5 and K19 (A) and K5 and K14 (B)
were subjected to dialysis against keratin assembly buffer (see
"Materials and Methods") for several hours. The K5-K19 filaments
obtained were slightly shorter and narrower than the K5-K14 ones.
Bar, 200 nm.
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Fig. 5.
Co-expression of type I and type II keratin
cDNAs in cultured BHK-21 cells. Cells were fixed and
double-immunofluorescence labelings were performed at 24-48 h after
transfection. The antigens detected are indicated in the upper
right corner of each micrograph. Frames A, A', and
A", co-expression of the K5-K19 pair led to the production
of aggregates containing both keratins. A" is a phase
contrast micrograph showing the morphology of the transfected cells
shown in A (see arrows). Frames B, B',
and C, co-expression of the K5-K14 pair gave rise to the
formation of a filament network, as shown in these two examples.
Frames D, D', and D", co-expression of K8-K19
produced filaments with a marked tendency to form bundles (again, the
phase contrast image of the transfected cell is shown in D"
(see arrow)). Frame E, co-expression of K8 and
K18 led to formation of a well extended array of thin filaments
throughout the cytoplasm. Frames F and F',
co-expression of K8-K14 led to a less well defined filamentous array.
Bar, A, C, D, and F, 27 µm;
B and E, 37 µm.
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Fig. 6.
Top, quantification of human keratin
levels in transfected BHK cells. Western blot analysis using anti-K14
(top) and anti-K19 (bottom) antibodies to detect
transfected keratin proteins. At 30 h after transfection, BHK
cells were scraped in loading buffer and total proteins resolved by
SDS-PAGE along with known amounts (10, 20, 40,and 60 ng) of recombinant
purified K19 or K14. Densitometric analysis indicates that similar
levels of K14 and K19 were expressed following transfection in BHK
cells. Bottom, electron microscopy of K19-transfected BHK
cells. Transfection of K5 and K19 cDNAs (A and
B) or of K19 cDNA alone (C and D)
into cultured BHK cells. Frames B and D are high
magnification micrographs of the regions boxed in frames A
and C, respectively. Immunogold labeling (10 nm,
arrows) with an anti-K19 antibody reveals the unpolymerized
nature of these K19-containing aggregates. Bars:A and
C, 1 µm; B and D, 200 nm.
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Fig. 7.
Transient expression of type I keratin
cDNAs in T51B-Ni and A431 epithelial cell lines. Cells were
fixed and double-immunofluorescence labelings were performed at 24-48
h after transfection. The antigens detected are indicated in the
upper right corner of each micrograph. Frames A
and A', T51B-Ni cells transfected with K19 and
double-stained for endogenous K14. Frames B and
B', T51B-Ni cells transfected with K18 and double-stained
for endogenous K14. Frames C and C", A431 cells
transfected with K19 and double-stained for endogenous K8-K18.
Frames D and D", A431 cells doubly-transfected
with the K14 and a green fluorescent protein (GFP) reporter
cDNA to unequivocally identify cells expressing the transfected
cDNAs (see "Materials and Methods"). In all instances the
transfected proteins integrated within the existing keratin IF network.
Bars, 15 µm.
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Fig. 8.
Transient expression of keratin cDNAs in
cultured PtK2 cells. Cultured epithelial PtK2 cells transiently
transfected with human keratin cDNAs. At 24-72 h
post-transfection, cells were fixed and processed for
double-immunofluorescence labeling. The antigens detected are indicated
in the upper right corner of each micrograph. Frames
A, A', B, and B', cells transfected with the K19
cDNA and double-stained for endogenous K8-K18. Frames C
and C, cells transfected with the K14 cDNA and
double-stained for endogenous K8-K18. Note that in contrast to K14,
expression of K19 results in aggregates disrupting the endogenous
network. Frames D and E, cells cotransfected with
the K8-K19 cDNAs (frame D) and K5-K19 cDNAs
(frame E). Co-expression of K8, but not K5, "rescues"
the disruption normally produced by K19 expression. Insets
show magnification of a portion of the transfected cell.
Bars are 50 µm; insets are 15 µm.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
ACKNOWLEDGEMENTS
FOOTNOTES
-ME, -mercaptoethanol; PCR, polymerase chain
reaction; BHK, baby hamster kidney; CMV, cytomegalovirus; BS3, bis-(sulfosuccinimidyl) suberate.
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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