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J. Biol. Chem., Vol. 276, Issue 37, 35123-35132, September 14, 2001
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From the Divisions of
Received for publication, April 13, 2001, and in revised form, June 5, 2001
The human type II hair keratin subfamily consists
of six individual members and can be divided into two groups. The group A members hHb1, hHb3, and hHb6 are structurally related, whereas group
C members hHb2, hHb4, and hHb5 are rather distinct. Specific antisera
against the individual hair keratins were used to establish the
two-dimensional catalog of human type II hair keratins. In this
catalog, hHb5 showed up as a series of isoelectric variants, well
separated from a lower, more acidic, and complex protein streak
containing isoelectric variants of hair keratins hHb1, hHb2, hHb3, and
hHb6. Both in situ hybridization and immunohistochemistry on anagen hair follicles showed that hHb5 and hHb2 defined early stages
of hair differentiation in the matrix (hHb5) and cuticle (hHb5 and
hHb2), respectively. Although cuticular differentiation proceeded
without the expression of further type II hair keratins, cortex cells
simultaneously expressed hHb1, hHb3, and hHb6 at an advanced stage of
differentiation. In contrast, hHb4, which is undetectable in hair
follicle extracts and sections, could be identified as the largest and
most alkaline member of this subfamily in cytoskeletal extracts of
dorsal tongue. This hair keratin was localized in the posterior
compartment of the tongue filiform papillae. Comparative
analysis of type II with the previously published type I hair keratin
expression profiles suggested specific, but more likely, random
keratin-pairing principles during trichocyte differentiation.
Finally, by combining the previously published type I hair keratin
catalog with the type II hair keratin catalog and integrating both into
the existing catalog of human epithelial keratins, we present a
two-dimensional compilation of the presently known human keratins.
The large keratin multigene family comprises the epithelial
keratins (also designated as "cytokeratins" or historically "soft keratins"), which are differentially expressed in the various types
of epithelia, and the hair keratins (historically designated as "hard
keratins"), which are involved in the formation of hard keratinized
structures such as hairs, nails, claws, etc. These keratins can be
divided into acidic type I and basicto-neutral type II members,
which form the 10-nm intermediate filament network of the cytoskeleton
of epithelial cells through the obligatory association of equimolar
amounts of type I and type II keratins (for review see Refs. 1-3).
Earlier gel electrophoretic studies on native hair keratins of several
mammals including man revealed the presence of four type I (44-48 kDa)
and four type II (55-60 kDa) members (4, 5). According to a
proposal by Heid et al. (4), hair keratins were collectively
designated "H" for hair, "b" for the basic members (Hb),1 and "a" for the
acidic members (Ha), with the two-dimensionally resolved and
Coomassie-stained protein spots of each subfamily being numbered from 1 to 4 in a counterclockwise manner. In addition to the "major" hair
keratins hHa1-hHa4 and hHb1-hHb4, a weakly expressed additional pair
was designated Hax/Hbx (4, 6). The hair keratin family was therefore
suggested to comprise 10 individual members and thus to be considerably
smaller than the family of epithelial keratins. Recent investigations
in our laboratory have shown, however, that the human hair keratin
family is distinctly more complex than previously assumed. Subsequent
to the characterization of seven type I hair keratins and four type II
hair keratins isolated from a human scalp cDNA library (7-11) and
the demonstration that the genes of type I and II hair keratins are
located on chromosomes 17q12-21 and 12q13, respectively (9), the
screening of a P1 artificial chromosome library with polymerase
chain reaction primers for two randomly selected type I hair keratins
yielded a 190-kilobase pair P1 artificial chromosome contig that
contained nine functional hair keratin genes, hHa1-hHa8
(including two highly related hHa3 genes, hHa3-I
and hHa3-II), and one transcribed pseudogene,
Recently, we used P1 artificial chromosome cloning to unravel the
organization of the human type II hair keratin gene locus. This
resulted in the characterization of an ~200-kilobase pair DNA domain
that also contained 10 hair keratin genes (14). However, unlike the
type I hair keratin genes, only six of these genes, hHb1-hHb6, were functional, whereas the remaining members
either represented nontranscribed ( Extraction of Hair Keratins, One- and Two-dimensional Gel
Electrophoresis, and Western Blot Procedure--
Plucked beard hairs
from two volunteers were freed from adhering outer and inner root
sheaths as described previously (4). The lower part of the follicles
was cut off and extracted for hair keratins (11). One-dimensional
SDS-polyacrylamide gel electrophoresis (PAGE, 10% polyacrylamide) and
two-dimensional resolution of hair keratins by isoelectric focusing
were carried out as reported previously (11, 13, 15). For isoelectric
focusing, the following mixture (total volume of 1 ml) of ampholines
(Bio-Rad) was used: pH 5-7, 400 µl; pH 4-6, 300 µl; and pH
3-10, 300 µl. One- and two-dimensional gels were stained with
Coomassie Blue. For Western blot procedures, the proteins separated on
gels were transferred to polyvinylidene difluoride membranes
(Immobilon-P, Millipore, Eschborn, Germany) using a semidry blotting
apparatus. After staining (0.1% Coomassie Brilliant Blue R250/50%
methanol), destaining (50% methanol), and blocking with 5% nonfat
milk powder in Tris-buffered saline, membranes were incubated with
primary antibodies (see "Antibodies" and Table I).
Peroxidase-coupled secondary antibodies (see "Antibodies") were
diluted 1:10,000 in 2.5% nonfat milk powder/0.1% Triton X-100
in Tris-buffered saline. Secondary antibodies were detected by enhanced
chemiluminescence (ECL, Amersham Pharmacia Biotech).
In Situ Hybridization (ISH) and Indirect Immunofluorescence
(IIF)--
ISH on cryostat sections of human scalp or plucked beard
hair follicles was carried out as described previously in detail (10,
11, 13). Human scalp samples, obtained from surgical interventions,
were provided kindly by Dr. Bernard Cribier (Dermatological Hospital,
Strasbourg, France). For the detection of the various hair keratin
mRNAs, the following probes were used (all probes were derived from
3'-untranslated regions and cloned into Bluescript KSII): hHb1
(phHb1-3'), a 400-bp PvuII/XhoI fragment; hHb2
(12.5cl2-3'), a 230-bp fragment obtained by the linearization of a
full-length hHb2 genomic clone by digestion with
BsrgI; hHb3 (phHb3-3'), a 520-bp
PstI/XhoI fragment; hHb4 (phHbX-3'), a 400-bp
BamHI fragment; hHb5 (phHb2-3'), a 657-bp
PvuII/XhoI fragment; and hHb6 (phHb4-3'), a
300-bp SmaI/XhoI fragment. The probes were
radiolabeled by in vitro transcription using
35S-rCTP.
For the recording of the ISH signals by reflection microscopy, a
confocal laser scanning microscope (LSM 510 UV, Carl Zeiss, Oberkochen,
Germany) was used. The instrument allows simultaneous visualization of
ISH in epi-illumination for the detection of reflection signals and
transmitted light in bright fields for hematoxylin staining. The two
signal channels were combined by an overlay in pseudocolor
(transmission image in green, electronically changed into black/white;
reflection image, i.e. ISH signals, in red).
IIF on cryostat sections of human scalp or plucked hair follicles was
carried out as described previously in detail (11, 13). The results
were documented with a photomicroscope (Axiophot 2, Carl Zeiss).
Antibodies--
Primary monoclonal antibodies against hHb1 (for
dilutions see Table I) and hHa2 (LHTric17 (IgM); IIF dilution 1:50)
both provided kindly by Dr. I. M. Leigh (Center for Cutaneous
Research, Royal London Hospital, London, UK). Antisera against the
remaining human type II hair keratins were produced in guinea pigs by
the injection of specific synthetic oligopeptides (100 µg/injection, see Table I), coupled to Keyhole limpet protein (Peptide Specialty Laboratories, Heidelberg, Germany). After the third booster injection, antisera against hHb2, hHb3, hHb4, hHb5, and hHb6 were obtained and
used at the dilutions indicated in Table
I. For ECL, peroxidase-coupled rabbit
anti-mouse or anti-guinea pig IgG (H+L) were used as secondary antibodies. For immunofluorescence, Cy2-, Cy3-, or Texas Red-coupled goat anti-mouse IgG or IgG+IgM or anti-guinea pig IgG were used at
dilutions of 1:50-1:200. All secondary antibodies were from Dianova
(Hamburg, Germany).
Sequence Characteristics of Human Type II Hair Keratins--
We
have shown previously that multiple sequence comparisons of the type II
hair keratins encoded by the six functional genes contained in the
human type II hair keratin gene domain on chromosome 12q13 led to a
conspicuous sorting into the two groups A and C (14, for the
designation of the two groups, see "Discussion"). Group A contained
the highly related hair keratins hHb1, hHb3, and hHb6, whereas group C
consisted of the less related hair keratins hHb2, hHb4, and hHb5. Group
A members possessed head domains of almost identical length (10, 14),
the sequence identities of which were nearly as high as those of the
rod domains. The tail domains were also rather conserved (Table
II), the lower homology value being
caused by sequence variations in the penultimate portion of the tail
domain (10, 14). In contrast, sequence identities of the group C
members were essentially restricted to the rod domains, for which
homology values were, however, distinctly lower than those determined
for the rod and even head domains of group A members (Table II).
Western Blot Analyses of Human Type II Hair Keratins--
With the
exception of the monoclonal hHb1 antibody, antisera were raised against
the various type II hair keratins by means of specific synthetic
peptides, which in most cases were derived from the structurally
variable tail domains (Table I). Total hair keratin extracts from human
hair clippings were separated by one-dimensional SDS-PAGE. The
Coomassie-stained patterns shown in Fig.
1, a and b,
lanes 1, consisted essentially of two protein doublets at
54-57 kDa (type II hair keratins) and 44-47 kDa (type I hair
keratins). In Western blots, the antisera against hHb1, hHb2, hHb3,
hHb5, and hHb6 each detected a single protein band within the type II
hair keratin region (Fig. 1a, lanes 2-6). In accordance with their calculated molecular masses (Table I), the
slightly smaller hair keratins hHb1, hHb3, and hHb6 (mean molecular
mass ~ 54,000 kDa) were obviously all contained in the lower
protein band (Fig. 1a, lanes 2, 4, and
6), whereas the larger hair keratins hHb2 and hHb5 (mean
molecular mass ~ 56,000 kDa) were constituents of the upper
protein band of the type II hair keratin pattern (Fig. 1a,
lanes 3 and 5). Remarkably, none of the antisera
raised against the four hHb4 peptides listed in Table I detected a
protein band when reacted with keratin extracts from clipped hairs
(shown for the antiserum against peptide 1 in Table I; Fig.
1a, lane 7). However, in cytoskeletal extracts of
human dorsal tongue comprising both epithelial and dermal tissue components (see the complex Coomassie-stained pattern in Fig. 1b, lane 2), this hHb4 antiserum clearly revealed
a distinct albeit weak protein band (Fig. 1b, lane
4). This protein was also detected in Western blots with the
antisera raised against hHb4 peptides 2-4 (Table I and results not
shown). In accordance with its calculated molecular mass (~65,000
kDa, Table I), the hHb4 protein runs distinctly above the hHb5 protein
(Fig. 1b, lane 3) detected in Western blots of
hair keratin extracts (Fig. 1b, lane 1).
The two-dimensional pattern of human hair keratins is shown in Fig.
2a. It can clearly be seen
that in the gel system used, the type II hair keratins were mainly
resolved into two major protein streaks, an upper one, resolved into
four visible protein spots in the more basic region, and a lower more
acidic one, which obviously consisted of more than one protein
(Fig. 2a). Slightly above the latter, a minor streak of
three faint protein spots could be detected (arrows in Fig.
2a). In Western blots, the hHb5 antiserum strongly reacted
with the upper four protein spots (Fig. 2b). Therefore,
every following blot with one of the remaining antisera was stripped
and re-exposed to the hHb5 antiserum, thus using hHb5 as a positional
reference. This strategy showed that the hHb1 antibody (Fig.
2c) and the antisera against hHb3 (Fig. 2e) and
hHb6 (Fig. 2f) uniformly detected the corresponding hair keratins within the major lower protein streak. Although the blots suggested that hHb3 was slightly more acidic than hHb1, hHb6 clearly represented the most acidic member of the three hair keratins (Fig.
2f). The hHb2 antiserum specifically reacted with the three minor protein spots above the bulk of hHb1, hHb3, and hHb6
(arrows in Fig. 2d).
To localize hHb4 relative to the other type II hair keratins, equal
amounts of the human hair and tongue extracts were mixed and resolved
two-dimensionally. Coomassie staining of the gel allowed the assignment
of the type II hair keratins hHb5 and hHb1, hHb3, hHb6, and the largely
unresolved bulk of type I hair keratins as well as the
differentiation-specific keratin pair K4/K13 of tongue epithelium (16).
Western blotting with the hHb4 antiserum against peptide 1 (Table I)
led to the occurrence of a single protein spot distinctly above hHb5
(designated by asterisks in Fig. 2h) in the
unstained area of the gel and showed that hHb4 represented the most
basic member of the type II hair keratin subfamily (Fig.
2h).
Expression of Type II Hair Keratins in the Human Anagen Hair
Follicle--
The expression patterns of the members of the two type
II hair keratin groups A and C in human scalp hair follicles were
investigated by both ISH using specific 3'-cRNA probes of the
respective hair keratin mRNAs and IIF by means of the specific
antibodies described above.
Group A Hair Keratins hHb1, hHb3, and hHb6 Are Expressed in the
Hair Cortex--
In line with a previous study in our laboratory (10),
ISH with the hHb1 and hHb3 cRNA probes on human scalp hairs revealed the expression of the respective mRNAs in the lower to mid-cortex region of the hair follicle (Fig. 3,
a and b). Previously we placed the onset of hHb6
mRNA expression slightly above that of hHb1 and hHb3 (10), but a
thorough analysis of multiple independent ISH with the hHb1, hHb3, and
hHb6 cRNAs rather suggested an almost simultaneous onset of expression
of the three mRNAs (Fig. 3, a-c). Unlike the hHb1 and
hHb3 transcripts, hHb6 transcripts clearly occurred up to the
keratogenous zone of the hair shaft (Fig. 3c). Indirect
immunofluorescence studies with the hHb1, hHb3, and hHb6 antibodies
confirmed the largely uniform onset of synthesis of the three hair
keratins in the lower to mid-cortex but showed that all of them could
be demonstrated up to the zone of fiber hardening (Fig. 3,
d-f). Remarkably, the hair follicle analyzed for hHb1
synthesis represented the rare case of a human scalp hair that
contained a medulla, in which hHb1 was also present (Fig.
3d). Consistently, the group A hair keratins were absent from the hair cuticle.
Expression Sites of Group C Hair Keratins hHb2, hHb5, and Hb4 in
the Hair Follicle Are Unrelated--
ISH with a specific hHb2 cRNA
probe on both a medullated beard hair (Fig.
4a) and human scalp hairs
(Fig. 4b) revealed transcripts of this hair keratin
exclusively in the single-layered hair cuticle, which can clearly be
distinguished from the cuticle of the inner root sheath at the light
microscopic level (Fig. 4a). hHb2 mRNA expression
started immediately above the line of Auber (17) and gradually vanished
at the height of the mid-cortex region (red arrows in Fig.
4, a and b). IIF studies with the hHb2 antiserum confirmed the restriction of this hair keratin to the hair cuticle (Fig. 5a and
inset), in which it occurred up to the zone of fiber hardening. Double-labeled IIF, using the hHb2 antiserum (red
in Fig. 5, b, b'), and the monoclonal antibody
against the previously described type I hair keratin hHa2 in the hair
cuticle (green in Fig. 5, b, b', and
Ref. 13) showed interesting details. First, the onset of synthesis of
the two cuticular hair keratins was different. Although hHa2 synthesis
clearly started below the line of Auber (green arrow and
green-colored hair cuticle cells in Fig. 5, b and
b') in the lower bulb region, that of hHb2 began distinctly
above this line (red arrows in Fig. 5, b and
b') thus leading to yellow-colored hHa2/hHb2 coexpressing
cells in the upper hair cuticle (Fig. 5, b and
b'). Second, unlike the strictly hair cuticle-specific hHb2
antiserum, the hHa2 antibody reacted, albeit faintly, also with matrix
and precortex cells (Fig. 5, b and b', and Ref.
13) but, if present, did not decorate cells of the central medulla
(Fig. 5b).
The hHb5 mRNAs occurred mainly in the matrix cells immediately
above the germinative cell compartment (Fig. 4, c and
d) and disappeared approximately at the same height of the
mid-cortex as the hHb2 mRNAs (compare Fig. 4, a,
b and c, and d). The cells bordering
the dermal papilla were devoid of hHb5 transcripts (white arrowheads in Fig. 4d), although occasionally some of
them appeared to be labeled (black arrowheads in
lower inset of Fig. 4d). A closer
inspection of the hybridization pattern suggested the presence of hHb5
transcripts also in the hair cuticle (Fig. 4d and
upper inset). Although the onset of cuticular and matricial
hHb5 mRNA expression was coincident, its cessation in the hair
cuticle occurred much earlier at the height of the precortex (red
double-headed arrows in Fig. 4d and upper
inset). IIF studies with the hHb5 antiserum confirmed this complex
expression pattern but in addition allowed the following of the fate of
cuticular and cortical hHb5 protein up to the keratogenous zone (Fig.
5, c and d). Moreover, double-labeled IIF using
the hHb5 antiserum (red in Fig. 5d) and the hHa2
antibody (green in Fig. 5d) not only supported
the notion that the onset of hair cuticle and matrix expression of hHb5
occurred at the same height in the lower bulb region of the follicle
(Fig. 5d) but also confirmed coexpression of both hair
keratins not only in matrix and precortex cells (compare Fig.
5b' and faint yellow staining in the matrix
region of the follicle in Fig. 5d) but also in the entire
hair cuticle (Fig. 5d). No hair keratin was detectable in
the lower matricial region, in which the progenitor cells of the
various compartments are assumed.
In line with the failure to demonstrate hHb4 protein in Western blots
of human hair keratin extracts (Fig. 1a), neither hHb4 transcripts nor hHb4 protein could be detected in human hair follicles by ISH or IIF (results not shown). However, both ISH and IIF on human
dorsal tongue sections clearly revealed a strong expression of hHb4
mRNAs (Fig. 6a) and
protein (Fig. 6b) in the suprabasal compartment of the
filiform papillae. Considering that in human tongue filiform papilla
units exhibit more than one of the typical hook-shaped spiny
protrusions, especially in the mid and posterior portion of the tongue
(18), it was difficult to cut cryostate sections in a way to obtain
strictly sagittal sections through the protruding spines. Because mouse
tongue filiform papilla units are preferentially organized into a
single spine (19, 20), the hHb4 antiserum was probed on longitudinal
sections of mouse tongue. As shown in Fig. 6, c and
c', the antiserum detected the murine ortholog of hHb4
specifically in their filiform papillae. A higher magnification of a
papilla unit combined with differential interference contrast
microscopy (Fig. 6c') not only allowed an accurate
distinction between the papilla unit and the adjacent interpapillary
tongue epithelium but also between the orthokeratinizing anterior
compartment and the hard keratinizing posterior compartment (ac and pc, respectively, in Fig. 6c')
of the papilla spine. It can be seen clearly that mHb4 is synthesized
at the base of the posterior compartment, and more precisely in a
cellular subcompartment directly apposed to the base of the adjacent
anterior compartment. Finally, mHb4 synthesis could also be
demonstrated in suprabasal cells of the parakeratinizing scale
epidermis mouse tail skin (Fig. 6d).
After the elucidation of the human type I hair keratin genes and
their expression patterns in the hair follicle as well as the assembly
of the encoded proteins into a catalog (13), the extension of these
studies to type II hair keratins became mandatory. The type I hair
keratin gene subfamily on chromosome 17q12-21 comprises nine genes,
which in terms of sequence homologies and gene organization can be
divided into three groups. Although two of these groups, A and B, each
encode highly related hair keratins, group C codes for less related
hair keratins (Ref. 12 and Fig. 7a). In contrast, the type lI
hair keratin gene subfamily located on chromosome 12q13 lacks
functional equivalents of type I group B genes, and its six members are
organized into type I group A and C counterparts, respectively (Ref. 14
and Fig. 7a). Compared with the selection of oligopeptides
used for the generation of specific antisera against the individual
type I hair keratins (13), we met with substantially less difficulties
in designing specific oligopeptides for the individual type II hair
keratins. Consequently, the individual antisera all detected only
single protein bands in one-dimensional Western blots of hair keratin extracts. Although we previously noticed considerable discrepancies between calculated molecular mass values and position in
one-dimensional gels for some members of the type I hair keratin
subfamily (13), the migration properties and mass calculations of type
II hair keratins agreed much better. The assignment of the type II hair keratins in Western blots of two-dimensionally resolved hair keratins was facilitated in that hair keratin hHb5, the largest and most alkaline member of the Coomassie-stainable proteins, could be used as
"marker" for the positioning of the remaining, distinctly more
acidic proteins. Of these, hHb1, hHb3, and hHb6 all showed similar
mobilities, whereas hHb2 was located slightly above the bulk of hHb1,
hHb3, and hHb6. Intriguingly, despite the use of antisera raised
against four different oligopeptides of the hair keratin hHb4, this
protein could not be detected in Western blots of hair keratin
extracts. However, in cytoskeletal extracts of human dorsal tongue,
i.e. another anatomical site known to express hair keratins
in its filiform papillae (21-23), all hHb4 antisera unambiguously
identified this hair keratin as the largest and most alkaline member of
the type II hair keratin family.
The catalog of human type II hair keratins resulting from
the compilation of these two-dimensional data is shown in Fig.
7b. A comparison of this catalog, in which gray
dots indicate the most alkaline isoelectric variants of the
various hair keratins, with the data proposed earlier by Heid et
al. (4) (Fig. 7c) reveals that none of the earlier
designations agree with the present ones. A priori, this can
not be expected because the previous nomenclature for type I and type
II hair keratins relies on a counterclockwise numerical designation of
Coomassie-stainable hair keratins (4), whereas the recent designation
is based on the immunodetection of defined hair keratin gene products. Similar to our study, one "minor" member of the type II hair
keratin subfamily, designated Hbx by Heid et al. (6) (Fig.
7c), could not be detected previously in Coomassie-stained
gels of human hair keratins or Western blots thereof using a pan type
II keratin antiserum (6). Hbx could, however, be revealed in
Coomassie-stained two-dimensional gels of cytoskeletal extracts of
isolated spines of bovine filiform tongue papillae (23). Here we have
demonstrated the identity of Hbx with hHb4 using two-dimensional
Western blots of human dorsal tongue cytoskeletal extracts with
specific hHb4 antisera (Fig. 2, g and h, see also
Fig. 7, b and c).
In Fig. 7d we have combined the type II hair keratin catalog
with the previously published type I hair keratin catalog (13) and
integrated both into the human type I and type II keratin catalog of
Moll et al. (16). As it stands, this human keratin catalog
now comprises 22 type II members (16 epithelial keratins and six hair
keratins) and 20 type I members (11 epithelial keratins and nine hair
keratins). Although we are fairly convinced that this catalog contains
the complete number of hair keratins (12, 14), there is strong evidence
that the number of keratins expressed in epithelia is far from being
complete. Recent estimates from the human genome sequencing project
predict the existence of as many as 111 keratin genes (30). A
compilation of all known type I and type II keratin genes/pseudogenes,
either published or present as EMBO/GenBank data base entries, amounts
to 82 genes (49 genes and 33 pseudogenes).2,3
Because these data might be slightly
underestimated,4 the
prediction of ~111 human keratin genes seems realistic, although the
number of functional genes will certainly be much lower.
The catalog of human keratins reveals a numerical imbalance between
type I and type II members. In the case of hair keratins, this is
mainly because of the lack of type II counterparts of the type I group
B hair keratin genes hHa7 and hHa8 (Fig.
7a). Both hair keratins show unusual expression restrictions
to central cortex cells of vellus hairs (hHa7) or single cortex cells
of terminal hairs (hHa8) (13). In addition, type I group B hair keratin
genes contain a transcribed pseudogene, To collectively describe the extremely complex expression pattern of
human type I and type II hair keratins in the hair follicle, we have
summarized the corresponding expression data schematically (Ref. 13 and
this paper) in Fig. 8, a and
b, for better understanding. For the sake of a comprehensive
interpretation of these data, in both schemes only mRNA expression
profiles of the various hair keratins are reported. To visualize the
sequential expression of the various hair keratins, the designations of
the individual type I (red) and type II (blue)
hair keratins in Fig. 8a stand for the site of onset of
their mRNA synthesis in the hair-forming compartments, with the
size of the letters reflecting the degree of expression. In Fig.
8b, vertical columns indicate the mRNA expression zones of the individual type I (red) and type II
(blue) hair keratins within the hair cuticle and
matrix/cortex, respectively. Because our IIF studies have shown that
independent of the onset of mRNA expression, virtually all hair
cuticle and matrix/cortex keratin proteins can be demonstrated up to
the zone of fiber hardening (thin vertical arrows in Fig.
8b), this implies that any hair cuticle cell leaving the
living cell compartment contains four different hair keratins, whereas
any cortex cell may accumulate an unprecedented number of up to 12 different hair keratins (Fig. 8b). In this respect, it is
highly astonishing that the impact of a disruptive mutation in a cortex
keratin, hHb1 or hHb6, is obviously not attenuated by the high number
of paralleling unaffected keratin pairings but entails such dramatic
alterations as the beaded-hair phenotype in Monilethrix patients (33,
34).
The Catalog of Human Hair Keratins
II. EXPRESSION OF THE SIX TYPE II MEMBERS IN THE HAIR FOLLICLE
AND THE COMBINED CATALOG OF HUMAN TYPE I AND II KERATINS*
§,, and Cell Biology and
¶ Tumor Cell Regulation, German Cancer Research Center,
Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
hHaA, within a 140-kilobase region (12). By means of both
specific cRNA probes and specific antibodies for the individual type I hair keratins, we subsequently elucidated the complex expression patterns of the members of this subfamily in the hair follicle (13). In
addition, the antibodies were used to establish by Western blotting a
two-dimensional catalog of the type I hair keratin subfamily (13).
hHbA,
hHbB, and hHbC) or transcribed (hHbD) pseudogenes. We were also able to show that the
type II hair keratin domain is flanked by the genes for the epithelial keratins K6hf and K7, respectively (14). In the present study, we have
once again used in situ hybridization with specific cRNA probes and indirect immunofluorescence studies with specific antibodies to determine the expression sites of the individual type II hair keratins in the human hair follicle. A comparison with the type I hair
keratin expression pattern suggests complex dynamics of hair keratin
pair formation during the process of hair shaft differentiation. We
also provide a two-dimensional catalog of the presently known human
type I and II hair keratins.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Type II hair keratins, calculated molecular mass values, synthetic
peptides used for the generation of antibodies/antisera, names of
antisera, and dilutions for ECL/IIF
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Amino acid sequence identities of human type II hair keratins
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Fig. 1.
Identification of human type II hair keratins
by SDS-PAGE and Western blot analysis. a, lane
1, Coomassie staining of total keratins from clipped hairs
transferred to a nylon membrane. The position of the collective type II
(HK Type II) and type I (HK Type I) hair keratins
are indicated. Western blots were performed with antisera against hHb1
(lane 2), hHb2 (lane 3), hHb3 (lane
4), hHb5 (lane 5), hHb6 (lane 6), and hHb4
(lane 7). b, Coomassie staining of total keratins
from clipped hairs (lane 1) and cytoskeletal extracts of
human tongue (lane 2). The Western procedure was performed
as shown in a by simultaneous exposure to antisera against
hHb5 and hHb4, which detect hHb5 in hair (lane 3) and hHb4
in tongue extracts (lane 4) (10% SDS-PAGE). Molecular
mass ranges (Mr) between 40 and 60 kDa are
indicated on the left-hand side of a and b.
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Fig. 2.
Identification of human type II hair keratins
by two-dimensional gel electrophoresis and Western blot analysis.
a, Coomassie staining of total hair keratins transferred to
a nylon membrane. HK Type II, type II hair keratins;
HK Type I, type I hair keratins. Western blots were
performed with the antiserum against hHb5 (b) or
successively with the antisera against hHb5 and hHb1 (c),
hHb2 (d, the arrows in d and
a indicate hHb2), hHb3 (e), and hHb6
(f). g, Coomassie staining of mixed cytoskeletal
extracts of human hair and tongue transferred to a nylon membrane. The
type II hair keratins hHb5, hHb1, 3, and 6, and the largely unresolved
type I hair keratins hHa1-6 (13) as well as the keratins K4 and K13,
prominent in tongue epithelium, are indicated. A, actin.
h, Western blot of g with the hHb4 antiserum. The
four asterisks indicate the positions of the isoelectric
variants of hHb5. The proteins were separated by isoelectric focusing
in the first dimension and by SDS-PAGE (SDS, 10% polyacrylamide) in
the second dimension.
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Fig. 3.
Demonstration of group A hair keratin
expression in hair follicles. ISH on cryostate sections of human
scalp with specific cRNA probes for mRNAs of hHb1 (a),
hHb3 (b), and hHb6 (c) are shown. IIF cryostate
sections of human scalp with specific antibodies/sera against hHb1
(d), hHb3 (e), and hHb6 (f) are also
shown. The red arrows in each panel demarcate the
zones of mRNA expression, and the green arrows in
d-f denote the zones of hair keratin proteins, with the
vertical arrows indicating the presence of proteins up to
the zone of fiber hardening. co, cortex; cu, hair
cuticle; irs, inner root sheath; ors, outer root
sheath; dp, dermal papilla; med, medulla. In
d-f, nuclei are stained with 4',6-diamino-2-phenylindole.
Bars, 200 µm.
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Fig. 4.
Demonstration of group C hair keratin
mRNA expression in hair follicles. ISH on cryostate sections
of (a), a plucked beard hair (b-d),
human scalp with specific cRNA probes for the mRNAs of hair keratin
hHb2 (a and b), and hHb5 (c and
d) are shown. The red arrows in each panel
indicate the zones of mRNA expression. The red double-headed
arrows in d and the upper inset denote the
cessation of hHb5 mRNA expression in the hair cuticle. The
white arrowheads in d indicate hHb5
mRNA-negative matrix cells lining the dermal papilla; some of these
cells, positive for hHb5 mRNAs, are marked by black
arrowheads in the lower inset of d.
au, line of Auber; icu, cuticle of inner root
sheath. For other designations, see the legend to Fig. 3.
Bars, 200 µm.
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Fig. 5.
Demonstration of group C hair keratin protein
expression in hair follicles. a, IIF on a human scalp
section with the specific hHb2 antiserum. The red arrows
indicate the zones of hHb2 mRNA expression in the hair cuticle (see
also Fig. 4, a and b). The inset shows
the labeled cuticle in a cross-section through the upper follicle.
b and b', double-labeled IIF with the hHb2
antiserum (red) and the hHa2 antibody (green).
The green arrows indicate the onset of cuticular hHa2
synthesis, the red arrows denote the onset of hHb2 synthesis
in the hair cuticle, the yellow designation a2+b2 in
b points to hHb2/hHa2 coexpression in the hair cuticle. Note
the weak hHa2 synthesis at the transition of the matrix (ma)
and lower cortex (lco) (b and b'),
which is absent in cells of the medulla (med)
(b). c, IIF on a human scalp section with the
specific hHb5 antiserum. The red arrows indicate the zone of
hHb5 mRNA expression in the cortex (see also Fig. 4, c
and d). The double-headed arrow denotes the site
of cessation of hHb5 mRNA expression in the hair cuticle. The
white arrowheads in the inset show
hHb5-expressing cells apposed to the dermal papilla. The green
vertical arrow indicates protein synthesis up to the zone of fiber
hardening. d, double-labeled IIF with the hHb5 antiserum
(red) and the hHa2 antibody (green). The
yellow-colored cells confirm synthesis of both hHb5 and hHa2
in the hair cuticle (see also Fig. 4d). The nuclei in
a, c, and d are stained with
4',6-diamino-2-phenylindole. For other designations, see the legend to
Fig. 3. Bars, 200 µm.
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Fig. 6.
Demonstration of hHb4 expression in the
filiform tongue papilla and the scale epidermis of mouse tail.
a, ISH with a specific cRNA probe for the hHb4 mRNA;
b, IIF with the hHb4 antiserum on cryostate sections of
human dorsal tongue. IIF with the hHb4 antiserum on cryostate sections
of mouse dorsal tongue (c and c') and mouse tail
skin (d) is shown. dp, dermal papillae;
bc, basal compartment; sc, suprabasal
compartment; pc, posterior compartment of the filiform
papilla; ac, anterior compartment of the filiform papilla;
ipc, interpapillary compartment; hsp, horny spine
of filiform papilla; isr, interscale of tail epidermis;
sr, scale region of tail epidermis. The dotted
lines in c and c' (differential interference
contrast and IIF) demarcate the border between the anterior and
posterior compartments of the filiform papilla. Bars, 200 µm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 7.
Schematic presentation of the human type I
and type II hair keratin loci and designation of gene groups
(a) and the catalog of human keratins
(b-d). a, note that both the size of the
genes and the intergenic distances are not drawn to scale (12, 14). The
shaded hHaA gene represents a transcribed
pseudogene (12, 32). b, two-dimensional catalog of human
type II hair keratins based on the identification of the individual
hair keratins by Western blots with specific antisera. Shaded
circles indicate the most alkaline isoelectric variant of each of
the hair keratins. The molecular masses of the proteins are indicated.
c, previous designation of type II hair keratins according
to Heid et al. (6). Note that the hatched hair keratin hHb4
in b (Hbx in c) is not detectable in keratin
extracts of hairs. d, combined human keratin catalog.
Epithelial keratins are indicated by a number (i.e. 1 = K1);
hair keratins by a letter and a number (i.e. a2 = hHa2).
Note that the two-dimensional position of the lately discovered
type I keratins expressed in the inner root sheath (28) are not known
and are not indicated in this figure. Abscissa, isoelectric
pH; ordinate, molecular mass values. 21,
the double circle indicates two keratins, K2e and K2p, that
exhibit highly similar migration properties in SDS-PAGE (24, 25).
62, the double circle indicates six K6
isoforms, K6a-f (26), as well as two K6-related keratins, K6hf (15)
and K6irs (cf. Ref. 27).
10/113, note that K11 is most probably a
polymorphic form of K10 that arises through the loss of glycine-rich
repeats in the head or tail domains (45). a14, in
5% of the human population, hHa1 occurs as a low molecular weight
isoform hHa1-T, which lacks the complete tail domain (11).
Superscripted numbers in this figure do not refer to footnotes.
hHaA (Fig.
7a), the mutated mRNA of which is, however, not
translated into a detectable protein (32). We were able recently to
show that both chimpanzees and gorillas possess functional
hHaA orthologs and that the human gene was inactivated
only recently, ~200,000 years ago during human evolution (32). More
importantly, not only the hair keratin encoded by the chimpanzee
ortholog of hHaA but also those encoded by the chimpanzee
orthologs of hHa7 and hHa8 were found to
constitute major components of cortex cells of terminal chimpanzee
hairs (32).5 These striking
differences between human and chimpanzee may indicate that since the
Homo-Pan divergence, the entire human type I
group B hair keratin gene cluster is under low evolutionary pressure and possibly on its way to being completely eliminated from the genome.
This fate might have already happened to its type II counterpart, from
which apparently only a nontranscribed pseudogene, hHbB, is left (see Fig. 7a).
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Fig. 8.
Schematic presentation of the complex pattern
of hair keratin expression in the human hair follicle. a,
schematic drawing of an anagen hair follicle in which the designations
of type I (red) and type II (blue) hair keratins
denote the onset of their mRNA expression. b, mRNA
expression zones of the major type I (red columns) and type
II (blue columns) hair keratins in the hair cuticle and the
matrix/cortex. mRNA expression profiles of minor hair keratins are
indicated by pink dotted columns. The numbers
1-7 demarcate "levels" of the hair follicle referred to
under "Discussion." The thin vertical arrows indicate
the presence of hair keratin protein up to the level of fiber
hardening. hHa21, besides the hair cuticle, this
protein is also weakly expressed in the upper matrix/lower cortex (see
Fig. 5, b and b', and Ref. 13).
hHa82, at present, this protein is only expressed in
single cortex cells (13). hHa73, at present, this
protein is only detectable in vellus hairs (13). Superscripted numbers
in this figure do not refer to footnotes.
It is evident that this complex scenario of hair keratin expression in the hair-forming compartments makes it difficult to clearly decipher the formation of specific type I and type II heteropolymeric hair keratin pairs as observed previously for keratins in various types of epithelia (1-3). The hair cuticle with its less complex expression pattern may serve as an example for this (Fig. 8b). In the lowermost portion of the cuticle (level 2 in Fig. 8b), the presence of two type I hair keratins hHa2 and hHa5 versus the presence of only one type II hair keratin, hHb5, clearly suggests a competition of hHa2 with hHa5 for filament formation with hHb5. Further up (level 3 in Fig. 8b), the type I hair keratins hHa2 and hHa5 are coexpressed temporarily with type II hair keratins hHb2 and hHb5 before hHa5 expression ceases and hence confronts hHa2 with hHb2 and hHb5 (level 4 in Fig. 8b), thus creating an inversion of the situation in the lowermost cuticle. Only in the uppermost region of active cuticular hair keratin expression is hHa2 left to specifically pair with hHb2 (level 5 in Fig. 8b). Collectively, this scenario, which resembles that of the complex epithelial keratin K1, K10, K9, and K2e expression pattern in suprabasal human plantar epidermis (35, 36), suggests competitive, complex, and unique type I and type II hair keratin-pairing patterns. The same dynamics hold true for the potential pairing possibilities of matrix keratins and in particular cortex keratins (Fig. 8b), the unprecedented complexity of which makes it difficult to assume the formation of specific pairs. It remains to be seen whether the assessment of the morphology (cf. 11) or viscosity of intermediate filaments assembled in vitro from various recombinant type I and type II hair keratins or biophysical investigations on affinity strengths between various in vitro combinations of type I and type II hair keratins (cf. 37) may be suitable approaches to ultimately solve the problem of specific or "promiscuous" hair keratin pairing in vivo.
It is also evident that the complex scenario of hair keratin expression
requires highly stringent control mechanisms at the gene level, in
particular if further studies should confirm specific rather than
random pairing patterns. At present, we know next to nothing about the
regulation of hair keratin expression in the anagen follicle. The only
regulatory factor for which the activation of hair keratin genes via
direct interaction with promoter elements has been demonstrated clearly
is the forkhead transcription factor Foxn1 (formerly Whn, winged helix
nude), the defective gene of which is responsible for the nude mouse
phenotype (38, 39). Recently, we gained evidence that HOXC13, up to now
the only member of the HOX family shown to produce a hair phenotype after mutant expression in mice (40), is not only able to bind to
specific TAAT binding motifs in hair keratin promoters but also to
strongly activate hair keratin gene expression in
vitro.6 A similar
function has been claimed for the lymphocyte enhancer factor 1, LEF-1
(41-43), the DNA binding consensus sequence CTTTGAA of which is
present in the promoters of almost all hair keratin genes (12, 14,
41). Recent transgenic experiments using a reporter gene under the
control of a sheep wool keratin promoter containing a mutated LEF-1
binding site suggested that LEF-1 may enhance transcriptions of hair
keratin genes by introducing changes in DNA conformation, thus allowing
other transcription factors to assemble into an active complex (42).
This view is contrasted by our own in vitro
transfection experiments, which clearly showed that the activation of
various hair keratin promotors by 
-catenin, generally thought to act
in combination with LEF-1 because of the absence of a DNA binding
domain, was completely independent of the presence or absence of the
LEF-1 binding site in the
promotors.7 It is evident
that these data represent only the tip of an iceberg and that the basic
control mechanisms governing hair keratin expression have yet to be
elucidated. In this context, it is worth mentioning that recent studies
in our laboratory have shown that the ordered hair keratin expression
seen in the hair follicle is largely maintained in pilomatricomas,
i.e. a tumor type thought to originate from the hair-forming
compartment of the hair follicle (Ref. 4 and references therein).
Therefore, pilomatricomas, and more importantly cell lines derived from
such tumors, should be suitable tools to address the compelling
question of how hair keratin gene expression is controlled.
| |
ACKNOWLEDGEMENTS |
|---|
We are grateful to Werner W. Franke for his continued interest in and support of this work and Harald Herrmann and Ilse Hofmann for helpful discussion. We thank Irene M. Leigh (London) for monoclonal hair keratin antibodies, Herbert Spring for help with confocal laser microscopy, and Ulrike Beckhaus for technical assistance.
| |
FOOTNOTES |
|---|
* This work was supported in part by Deutsche Forschungsgemeinschaft Grant Schw 539/4-1. This is Paper II in the series "Catalog of Human Hair Keratins." Ref. 46 is Paper I in the series.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.
§ To whom correspondence should be addressed: German Cancer Research Center, Division of Cell Biology, A0100, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. Tel.: 49-6221-42-3436; Fax: 49-6221-42-3404; E-mail: Langbein@dkfz.de.
Published, JBC Papers in Press, July 9, 2001, DOI 10.1074/jbc.M103305200
2
At present, 24 human type I keratin
genes/pseudogenes (14 epithelial keratin genes
(K9-K20,3 hIRSa-1,
hIRSa3-1 (28), and K23 (29)) and 10 hair keratin genes/pseudogenes (hHa1, hHa2, hHa3-I,
hHa3-II, hHa4, hHa5, hHa6, hHa7, hHa8, and hHaA (12)) have
been characterized. In addition, there is evidence for four further
genes next to hIRSa-1 and hIRSa3-1 (28) and for
two pseudogenes of each K14, K16, and K17, as well as one K19
pseudogene.3 Moreover, up to 20 pseudogenes have been
suggested for K18.3 This would bring the total number of
type I keratin genes/pseudogenes to 55. Similarly, 27 human type II
keratin genes/pseudogenes (17 keratin genes/pseudogenes (K1,
K2e, K2p, K3, K4,
K5, K6a-f, K6hf, K6irs,
K7, K8, and K8)3 and 10 hair keratin genes/pseudogenes (hHb1, hHb2,
hHb3, hHb4, hHb5, hHb6,
hHbA, hHbB, hHbC, and
hHbD (14)) are known, thus making a total of 82 keratin
genes/pseudogenes.
3 M. A. Rogers, manuscript in preparation.
4 Of the 27 type II keratin genes, 13 are located on a 240-kilobase DNA contig (14). Furthermore, a YAC clone, 630 kilobases in size and containing the known type II epithelial keratin genes, has been reported (31). If a comparable gene density on the remaining 390 kilobases of the YAC contig exists as for the 240-kilobase contig, 21 instead of 14 genes should be expected.
5 L. Langbein, H. Winter, M. A. Rogers, S. Praetzel, and J. Schweizer, manuscript in preparation.
6 L. F. Jave-Suarez, H. Winter, L. Langbein, M. A. Rogers, and J. Schweizer, manuscript in preparation.
7 L. F. Jave-Suarez, H. Winter, L. Langbein, M. A. Rogers, and J. Schweizer, unpublished data.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: Hb, basic hair keratins; Ha, acidic hair keratins; PAGE, polyacrylamide gel electrophoresis; ISH, in situ hybridization; IIF, indirect immunofluorescence; bp, base pair.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
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 J. Gotter, B. Brors, M. Hergenhahn, and B. Kyewski Medullary Epithelial Cells of the Human Thymus Express a Highly Diverse Selection of Tissue-specific Genes Colocalized in Chromosomal Clusters J. Exp. Med., January 20, 2004; 199(2): 155 - 166. [Abstract] [Full Text] [PDF]
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 F. J. A. Conti, R. J. Rudling, A. Robson, and K. M. Hodivala-Dilke {alpha}3{beta}1-integrin regulates hair follicle but not interfollicular morphogenesis in adult epidermis J. Cell Sci., July 1, 2003; 116(13): 2737 - 2747. [Abstract] [Full Text] [PDF]
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M. A. Rogers, L. Langbein, H. Winter, C. Ehmann, S. Praetzel, and J. Schweizer Characterization of a First Domain of Human High Glycine-Tyrosine and High Sulfur Keratin-associated Protein (KAP) Genes on Chromosome 21q22.1 J. Biol. Chem., December 6, 2002; 277(50): 48993 - 49002. [Abstract] [Full Text] [PDF]
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Y. Shimomura, N. Aoki, J. Schweizer, L. Langbein, M. A. Rogers, H. Winter, and M. Ito Polymorphisms in the Human High Sulfur Hair Keratin-associated Protein 1, KAP1, Gene Family J. Biol. Chem., November 15, 2002; 277(47): 45493 - 45501. [Abstract] [Full Text] [PDF]
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 V. Vegesna, J. O'Kelly, M. Uskokovic, J. Said, N. Lemp, T. Saitoh, T. Ikezoe, L. Binderup, and H. P. Koeffler Vitamin D3 Analogs Stimulate Hair Growth in Nude Mice Endocrinology, November 1, 2002; 143(11): 4389 - 4396. [Abstract] [Full Text] [PDF]
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L. F. Jave-Suarez, H. Winter, L. Langbein, M. A. Rogers, and J. Schweizer HOXC13 Is Involved in the Regulation of Human Hair Keratin Gene Expression J. Biol. Chem., January 25, 2002; 277(5): 3718 - 3726. [Abstract] [Full Text] [PDF]
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