close

Вход

Забыли?

вход по аккаунту

?

Tenascin localization in skin wounds of the adult newt Notophthalmus viridescens.

код для вставкиСкачать
THE ANATOMICAL RECORD 230:451-459 (1991)
Tenascin Localization in Skin Wounds of the Adult
Newt Nofophthalmus viridescens
DONALD J. DONALDSON, JAMES T. MAHAN, HUI YANG, AND
KATHRYN L. CROSSIN
Department of Anatomy and Neurobiology, University of Tennessee Center for the Health
Sciences, Memphis, Tennessee (D.J.D.,J.T.M., Y.H.); The Rockefeller University,
New York, New York 10021 (K.L.C.)
ABSTRACT
Earlier studies have shown that the extracellular matrix (ECM)
protein tenascin (TN) is present between uninjured epidermal cells of urodele
appendages, but is absent from most of the mesenchymally derived ECM. Following appendage amputation, this distribution is reversed. TN is lost from the epidermis and appears in the ECM of the stump and the regeneration blastema. In the
present study, monoclonal and polyclonal antibodies to TN were used to localize
this protein immunohistochemically in limbs of the adult urodele Notophthalmus
viridescens a t various stages following skin removal with or without damage to
underlying muscle to determine 1)if the loss of TN by the epidermis and its gain
by mesenchymal tissues occurs in wounds that do not require regulation by epigenetic mechanisms, and 2) if TN is present in the provisional wound matrix
beneath migrating epidermal cells. In addition, skin explants were cultured on
TN-coated dishes to learn if TN possesses active sites that can support epidermal
cell migration. The results indicate that simple wounding leads to the same TN
patterns a s occurs following limb amputation. Tenascin loss from the epidermis
could be seen a s early a s 6 h r after wounding, a time during which migrating
epidermal cells are moving over the wound bed. During this period, there was no
evidence of TN in the provisional wound matrix. In contrast to collagen, which
supports considerable epidermal cell migration from skin explants, TN allowed no
more migration than did the inactive protein, myoglobin.
The ability of epidermal cells to engage in the coordinated movements required for wound closure is dependent on the presence of appropriate macromolecules
in the provisional extracellular wound matrix. Known
components of this matrix include fibrinogen and fibronectin (Clark e t al., 1982), proteins that are able to
mediate newt epidermal cell migration through interaction with epidermal receptors capable of recognizing
the amino acid attachment sequence, Arg-Gly-AspSER (RGDS) (Donaldson et al., 1987). Newt epidermal
cells are also able to migrate on the ECM proteins,
collagen, and vitronectin through the same or similar
receptors a s those involved in migration over fibrinogen and fibronectin (Donaldson et al., 1987). Migration
does not occur, however, on a variety of other proteins
such a s casein, myoglobin, fetuin, and BSA (Donaldson
and Mahan, 1984). While there is evidence that newt
epidermal cells do not require the RGD signal to migrate (Donaldson et al., 1988), the presence of such a
sequence in a given protein suggests that it might have
epidermal migration-supporting capacity. Tenascin
(TN), a recently discovered extracellular matrix protein, falls into this category.
Known variously as myotendinous antigen (Chiquet
and Fambrough, 1984a,b), glioma mesenchymal extracellular matrix protein (Bourdon et al., 1985), cytotactin (Grumet et al., 19851, J1 (Kruse et al., 1985), and
hexabrachion (Erickson and Iglesias, 1984), tenascin
(c)
1991 WILEY-LISS, INC
(Chiquet-Ehrismann e t al., 1986) is a high molecular
weight glycoprotein with six apparently identical
RGD-containing subunits, each of which forms one of
its six arms (see review by Erickson and Bourdon,
1989). The expression of TN in developing tissues is so
spatially and temporally regulated that i t is believed to
play a n important role in morphogenetic events (Tan et
al., 1987). Tenascin is common in mesenchymal tumors
and carcinomas and may be present even when it is
absent from the normal tissue of origin (Mackie et al.,
1987). Though i t is largely absent from unwounded
adult rat skin, TN rapidly appears in the dermis adjacent to a wound and in the granulation tissue that
develops later (Mackie et al., 1988). The fact that forming wound epithelium was always underlain by TN in
these latter studies prompted the authors to suggest
that TN might somehow facilitate epidermal cell migration.
Two studies have described the immunolocalization
of TN in normal and regenerating urodele appendages.
In the mesenchymal tissues of normal adult newt limbs
Received September 25, 1990; accepted February 4, 1991.
Address reprint requests to Donald J. Donaldson, Department of
Anatomy and Neurobiology, University of Tennessee Center for the
Health Sciences, Memphis, TN 38163.
452
D.J. DONALIISON ET AL.
the molecular weight of intact TN in immunoblots of
blastemal extracts, and both stain this same protein in
MT1 immunoprecipitates from blastemal extracts
(Onda et al., 1990). As a control for MT1 staining, we
utilized MC-480, a n IgM monoclonal directed against
the SSEA-1 carbohydrate epitope on glycolipids and
glycoproteins in mouse embryos and embryonal carcinomas (obtained from the Developmental Studies Hybridoma Bank a t Johns Hopkins School of Medicine).
In addition to MT1, two different polyclonal preparations were used to localize TN in normal skin and 24
h r wound epithelium. The one used in Figure 6 was a
rabbit anti-chick brain cytotactin (TN) serum previously described and characterized in Hoffman et al.
(1988). Identical results were obtained with a guinea
pig anti-chick TN serum (0-45% ammonium sulfate
cut dialyzed against PBS), prepared by the same
method as the rabbit anti-TN described in ChiquetEhrismann et al. (1986) (a kind gift from Dr. Eleanor
Mackie). Nonimmune rabbit serum and nonimmune
guinea pig serum (0-45% ammonium sulfate cut dialyzed against PBS) were used as controls.
To prepare a polyclonal antibody against newt fibronectin (FN), FN purified from newt plasma by affinity
chromatography on gelatin-agarose (Sigma) was subjected to preparative SDS polyacrylamide gel electrophoresis under reducing conditions. The region of the
gel containing FN (220 kD) was excised, emulsified in
adjuvant, and used for rabbit immunization. Anti-newt
FN IgG and preimmune IgG were purified by protein-A
chromatography. Both anti-newt FN IgG and preimmune IgG were absorbed with FN-depleted newt
MATERIALS AND METHODS
plasma to increase specificity. After absorption, antiAnimals
FN IgG stained two bands in immunoblots of newt
Adult male newts (Notophthalmus uiridescens) were plasma, both of which comigrated with corresponding
purchased from the Connecticut Valley Biological Sup- bands in human FN standards.
ply Co., Southampton, MA. Details of animal maintelmmunocytochemistry
nance have been described previously (Donaldson and
For TN localization, cryostat sections of newt tissue
Mahan, 1983).
were incubated in MT1 (approx. 90 Fg of immunoglobExperimental Wounds
uliniml of PBS), a t room temperature (rm temp) for 1
Under general anesthesia produced by whole animal h r followed by three washes in 0.05% Triton/PBS. After
immersion in 0.15% 3-aminobenzoic acid ethyl ester a n additional PBS wash, the sections were exposed to
(MS 222, Sigma Chemical Co., St. Louis, MO), a full anti-mouse immunoglobulin conjugated to Texas Red
thickness rectangle (1.5 x 3 mm) of skin was removed (Vector Labs, Burlingame, CA) in PBS for 1 h r a t rm
from the dorsal surface of each hind limb between the temp. Sections were then washed again and coverknee and ankle (type I wounds). Usually, the superfi- slipped with glycerol/PBS (3:l) containing 2.5% 1,4cial fibers of the underlying skeletal muscle were then diazabicyclo [2.2.2] octane (Sigma) to retard bleaching.
Polyclonal antisera against TN were used at 1:lO
minced with iridectomy scissors (type I1 wounds). Animals were placed in tenth strength Holtfreter’s solu- and 1 5 0 dilutions in PBS containing 1% BSA for 1 h r
tion (an amphibian saline) containing streptomycin a t rm temp. Following three PBS washes, the sections
sesquisulfate (Sigma) at a final concentration of 5 mg/L were incubated for 1 h r at rm temp in either goat antito allow healing. For immunocytochemistry, sections guinea pig or anti-rabbit IgG, both of which were confrom at least four animals were examined for each con- jugated to Texas Red (Vector). Washing and coverslipping were as described above.
dition a s indicated in the figure legends.
To localize FN, cryostat sections or whole mounts of
Primary Antibodies
wounds that had not yet formed a wound epithelium
Tenascin was localized through the use of MT1, a were treated overnight a t 4°C with polyclonal anti-FN
monoclonal IgM kindly provided by Dr. Roy Tassava, IgG that had been absorbed with FN-depleted plasma.
Ohio State University. Made against homogenates of Bound antibodies were visualized by exposure of the
newt limb regeneration blastemas, MT1 appears to be treated tissue sequentially to avidin- and biotindirected against newt TN by a variety of criteria. Thus, blocking solutions, biotin-labeled guinea pig antia polyclonal antibody against chick TN competes effec- rabbit IgG, and avidin Rhodamine (all from Vector).
tively for MT1 binding sites in sections of regeneration Whole mounts were cryosectioned and all sections were
blastemas, the two antibodies both stain a protein with washed and coverslipped a s above.
(Onda et al., 1990) and the normal tail of larval Pleurodeles waltz (Arsanto et al., 1990), TN was restricted
to discrete locations such as tendons, ligaments, myotendinous junctions, periosteum, and perichondrium.
Most of the extracellular matrix (ECM), however, was
TN-negative. Surprisingly, TN was also found in the
epidermis where it appeared to be in the intercellular
space between keratinocytes. Within a few days following amputation of either appendage, there was widespread acquisition of TN by previously negative stump
connective tissue. The ECM of regeneration blastemas
was likewise strongly TN-positive. Wound epithelium,
however, showed a great reduction in TN such that on
newt limbs TN was absent a t 1 day post-amputation
(the earliest stage examined) and thereafter was only
marginally present until digit differentiation began.
On tails, wound epithelium was TN-negative from day
4 (the earliest stage for which data was presented)
through day 14.
In the present study, we have sought to determine if
the apparent loss of TN in newt wound epithelium and
the acquisition of TN by mesenchymal tissue can occur
in response to wounds that do not involve amputation.
In so doing, we were particularly interested in localizing TN during the migratory stage of wound closure [a
stage not examined by either Onda et al. (1990) or Arsanto et al. (199011 to learn whether this protein is a
component of the provisional wound matrix, where it
might function a s a substrate for translocating epidermal cells. We also tested purified TN directly for its
ability to support newt epidermal cell migration.
453
T E N A S C I N AND SKIN WOUNDS
Skin Explants Cultured on TN
Tissue culture dishes were coated with the indicated
amounts of either bovine type I collagen (obtained from
Dr. Mustafa Dabbous, Dept. of Biochemistry, University of Tennessee, Memphis, TN), equine myoglobin
(Sigma), or human TN (Telios Pharmaceuticals, San
Diego, CA). Ten microliter aliquots of test proteins
were placed in 50 mm2 circles (six per dish) and allowed to dry overnight at 23°C. Before use, the dishes
were washed four times with distilled water. The collagen was made up in 0.1 M acetic acid; myoglobin and
TN were in PBS. Pieces of full-thickness newt skin, as
described earlier under Experimental Wounds, were
then explanted onto the coated spots so that for each
explant placed on myoglobin or TN a control explant
from the contralateral limb was placed on collagen.
Five milliliters of 60% CEM culture medium (B&B/
Scott Labs., Fiskeville, RI) was then added to each dish.
After 16 h r incubation a t 23"C, the explants were fixed
in 10% formalin. The amount of migration in each explant was determined planimetrically as described previously (Donaldson et al., 1987). To compare the migratory performance on each experimental substrate to
the performance on collagen, the following formula was
used:
planimeter value for an individual explant
group mean for the appropriate contralateral collagen controls
x 100.
These calculated values were then used to generate
the means shown in Figure 7.
RESULTS
TN Localization in Normal Limbs Using the
Monoclonal, MTl
Newt skin consists of a stratified squamous epithelium four or five cells thick resting on a connective
tissue dermis that contains large oval mucous glands
and numerous melanocytes. In the dorsal part of the
hind limbs (where these experiments were conducted),
the dermis, in turn, rests on the limb musculature (inset in Fig. 1).In samples from unwounded limbs (skin
plus a small amount of superficial muscle), MT1 reactivity was limited to tendons, portions of the interface
between glands and the adjacent tissue, and, most significantly for this study, the epidermis. Within the epidermis, MT1 immunoreactivity was punctate and appeared to be localized in the intercellular space where
it surrounded the apical ends of basal cells and often
completely enclosed cells in the second and third layers. In some areas, positive fluorescence was also seen
along the dermo-epidermal junction (Fig. 1).Dermal
connective tissue, gland cells, and underlying skeletal
muscle were negative. No fluorescence was detected in
control sections incubated with a n equivalent concentration of MC-480 (a monoclonal antibody that, like
MT1, is of the IgM isotype).
TN Localization in Wounded Limbs Using MT1
Within 6 h r after limbs were inflicted with type I1
wounds (the earliest time point sampled), TN immunoreactivity in the epidermis immediately adjacent to
the wound was much reduced. This can be seen in Figure 2, which shows a segment of epidermis (approxi-
mately 1 mm from the wound) in which only scattered
particulate fluorescence remains. No TN immunofluorescence was detected in the forming wound epithelium
(Fig. 3). Nor did we see any evidence of TN on the
wound bed (arrow in Fig. 3). This is in contrast to fibronectin (FN) staining which could be seen on wound
beds when a polyclonal anti-FN was used to treat either tissue sections or dissected wounds which were
sectioned after antibody exposure. This is shown in the
inset in Figure 3 where anti-FN treatment prior to
sectioning has produced a continuous immunoreactive
band on the wound bed. Absorption of anti-FN with
purified newt FN abolished the staining. Presumably,
if FN is available to antibodies, i t is also available to
migrating cells. To our knowledge, this is the first direct evidence in any species that wound-associated FN
is actually exposed on the wound bed.
After 24 hr, type I1 wounds were completely closed by
a multilayered wound epithelium. Under the wound
epithelium, injured muscle fibers were beginning to
break down (Fig. 4A). Tenascin appeared to be absent
from the epidermis bordering the wound (Fig. 4B) and
from the wound epithelium (Fig. 4C). Some TN immunolabeling was observed in the deeper tissues but this
was limited to lengths of tendon, which are positive
even before wounding, and infrequent accumulations
of what appeared to be a vacuolated exudate (Fig. 4C).
In the ensuing days and weeks, injured muscle fibers
degenerated and were replaced by a highly cellular
wound matrix that stained heavily for TN (Fig. 5). No
labeling of this matrix was seen when the control
monoclonal, MC-480, was used in place of MT1. Even
after 3 weeks, there was little if any TN immunoreactivity in either the epidermis immediately bordering
the wound (Fig. 5B) or in the wound epithelium (Fig.
5C). Since the experiment was not carried beyond 3
weeks, we do not know when normal epidermal TN
reactivity reappears.
Tenascin also appeared under the wound epithelium
in type I wounds where the skin was removed without
damage to underlying muscle. Surprisingly, in addition to finding TN in the reforming dermis, we also
found it around the more superficial muscle fibers under the wound (not shown). The epidermal pattern was
identical to that described above for type I1 wounds.
TN Localization in Wounded Limbs Using
Polyclonal Anti- TN
To determine if the loss of immunoreactivity detected with MT1 was limited to the MT1 epitope, we
stained 24 h r wound epithelium with two different
polyclonal antisera against chick TN. Each antiserum
produced the result shown in Figure 6 where the 24 h r
wound epithelium (we) is essentially devoid of stain
while the epidermis from the contralateral unwounded
limb (ne) shows the typical TN staining pattern of normal skin. Thus, the reduction in TN immunolabeling
cannot be explained by the loss or masking of a single
epitope, but probably represents a n effect involving the
entire TN molecule.
TN as a Migration Substrate
A major goal of this study was to determine if TN
might play a role in wound closure by acting a s a substrate for epidermal cell migration. Our immunohisto-
454
D.J. DONALDSON ET AL.
Figs. 1-3
TENASCIN AND SKIN WOUNDS
455
chemical results showing loss of TN immunoreactivity Arsanto et al. (1990) in which TN was found in uninin migrating epidermis could mean that TN is not jured urodele epidermis, and the findings of Mackie e t
present in the wound environment during the time al. (1988), in which the appearance of TN in wounded
when a wound epithelium is forming. If this is true, rat skin correlated both temporally and spatially with
then epidermal cells clearly do not accomplish wound migration of epidermal cells during wound closure,
closure by migrating on this protein. It may be, how- suggested that TN might play a n important role in
ever, that the amount of TN present in the wound en- formation of a wound epithelium in urodeles. One way
vironment had simply fallen below our level of detec- it could do this would be by functioning directly a s a
tion. We therefore decided to test TN directly for its substrate for keratinocyte migration. Information on
ability to support migration. Pieces of newt skin were the likelihood of this possibility was obtained by imexplanted onto plastic dishes coated with collagen (a munohistochemical localization of TN in normal and
positive control), myoglobin (a negative control), or TN wounded newt limbs.
In unwounded newt skin, TN immunoreactivity, a s
and were then incubated in culture medium for 16 hr.
In this system, proteins like collagen support the for- determined with the MT1 monoclonal, is found primamation of a considerable halo of migrated epidermal rily a s punctate deposits that appear to be between
cells around the explant in the time allowed. Unlike epidermal cells. Following removal of a small piece of
the robust response to collagen, migration on TN was skin with or without damage to underlying muscle,
not significantly different from migration on the neg- there is a relatively rapid loss of TN staining, suggestative control, myoglobin (Fig. 7). This failure of TN to ing that the epidermal cells composing the wound epsupport migration in a direct trial and the immunohis- ithelium contain little if any TN. Nor is there any detochemical evidence discussed above is consistent with tectable TN on the surface over which the wound
the idea that TN is not a migration-supporting compo- epithelium migrates, suggesting that TN is not a major
nent of the provisional wound matrix during epidermal component of the provisional wound matrix.
The monoclonal antibody that we used in this study
closure.
recognizes TN in its native but not in its reduced form
DISCUSSION
(Onda et al., 1990), which suggests that the epitope
Tenascin is unique among extracellular matrix mol- recognized is in the disulfide-rich central region of the
ecules in that i t possesses both a cell binding site and molecule, where the six arms meet (Erickson and Boura n anti-adhesive region for certain cells (Spring et al., don, 1989). Had we limited our localization experi1989). The adhesive site resides in the distal region of ments to the use of MT1, i t could be argued t h a t i t was
each of the six arms that surround the central core of not the entire TN molecule that was lost from migratthe molecule (Friedlander e t al., 1988; Spring et al., ing epidermal cells, but only a portion containing the
1989).Most cells that adhere to TN either do not spread MT1 epitope. Or the epitope in question may have
(Chiquet-Ehrismann et al., 1988) or spread less com- somehow become masked. The fact t h a t wound epithepletely on i t than they do on substrates such as fibro- lial reactivity to two different polyclonal anti-TN sera,
nectin (Bourdon and Ruoslahti, 1989). However, quail which presumably are directed against multiple
neural crest cells not only adhere to TN but also effec- epitopes, was also lost weakens the above arguments.
tively migrate on it (Halfter et al., 1989). These obser- We think that loss of immunoreactivity is most likely
vations coupled with those of Onda et al. (1990) and due to proteolytic loss of either a large immunogenic
region or loss of the entire TN molecule rather than
masking of all available immunogenic epitopes. The
localization data therefore suggest that TN does not
function as a keratinocyte migration substrate.
The apparent lack of TN along the migratory path of
(Figs. 1-3) Fig. 1. Cryostat section of newt skin and underlying
muscle from an unwounded limb stained with a monoclonal antibody keratinocytes is in distinct contrast to fibronectin,
to TN. The inset shows a section through a similar region stained
which forms a thin coating over the wound bed and
with hematoxylin and eosin (H & E). Tenascin is found only in the
which, by whole mount staining with anti-fibronectin,
intercellular spaces between epidermal cells, in certain regions of the
appears to be available to epidermal ECM receptors. It
dermo-epidermal junction, and in portions of the interface between
glands and dermal connective tissue. g = glands, m = muscle. The
is likely that other proteins such as fibrinogen, collaarrows point to the dermo-epidermal junction. Magnification of main
gen, and vitronectin also play roles which vary in impanel, 162 x ; inset, 50 x .
portance from case to case depending on the extent to
which plasma proteins are deposited on the wound bed.
Fig. 2. Cryostat section through uninjured skin and underlying
muscle from a region adjacent to a 6 hr type I1 wound stained with a
Since there is no detectable TN among the epidermal
monoclonal antibody to TN. Note how TN staining in the epidermis cells of adult rats (Mackie et al., 1988) or adult humans
has been reduced from the extensive pattern in Figure 1 to scattered
(Lightner et al., 1989), it is somewhat surprising and
dots. m muscle. The arrows point to the dermo-epidermal junction.
interesting that normal unwounded urodele epidermis
162 x .
contains a n abundance of TN. The function of this epiFig. 3. Cryostat section through the leading edge of the forming
dermal TN is unknown. Its adhesive properties suggest
wound epithelium in a 6 hr type I1 wound stained with a monoclonal
that it may simply be there as a n intercellular cement.
antibody to TN. The rectangle in the upper inset ( a section stained
Possessing a s it does six subunits, it has the necessary
with H & E ) shows the region pictured in the main panel. The dotted
line in the main panel outlines the advancing wound epithelium.
multiplicity of binding sites to join adjacent cells. The
Note that TN staining is absent from both the migrating epidermis
rapid loss of TN following wounding could explain why
and the wound bed (arrow). The lower inset shows a thin band of
migrating epidermal cells are able to change their relfibronectin on the wound bed revealed by exposure of a fresh type I
ative positions in the sheet a s a wound epithelium
wound to fibronectin antibodies before sectioning. m = muscle. Magnification of main panel, 162 x ; upper inset, 45 x , lower inset, 78 x .
forms (Mahan and Donaldson, 1986; Repesh and Ober:
456
D.J. DONALDSON ET AL.
Figs. 4-5
457
TENASCIN AND SKIN WOUNDS
(Figs.4, 5) Fig. 4. Cryostat sections through skin and underlying
tissues from a sample taken 24 hr post-wounding (type I1 wound).
Panel A is a section stained with H & E. The lower two panels show
the results of staining an adjacent section with a monoclonal antibody
to TN. In panel A, the wound is to the right of the dotted line. The
rectangles on the left and right show the approximate locations of the
regions depicted in panels B and C, respectively. In panel B, note
that all TN immunoreactivity has disappeared from the uninjured
epidermis (ue) adjacent to the wound. In panel C, there is likewise no
TN evident in the wound epithelium (we). Deep to the wound surface,
occasional sections included immunolabeled tendon (t), which is positive even in unwounded limbs. Infrequently, localized areas of a vacuolated material (v) that appeared to contain TN were also seen deep
to the wound epithelium. Otherwise, there was no labeling of the deep
tissues. The arrows in both lower panels point to the dermo-epidermal
junction. Magnification of A, 66 x ; B,108 x ; C, 108x .
Fig. 5. Cryostat sections through skin and underlying tissues from
a sample taken 3 weeks after wounding (type I1 wound). Panel A is a
section stained with H & E. The two lower panels show the results of
staining an adjacent section with a monoclonal antibody to TN. In
panel A, the left and right rectangles show the approximate locations
of the regions depicted in panels B and C, respectively. Though the
exact boundaries of the original wound are difficult to determine this
long after wounding, the presence of glands and intact muscle under
the epidermis enclosed by the left rectangle indicates that this represents an area adjacent to the original wound. Panel B shows that the
only TN staining here is in a length of tendon (t). Uninjured epidermis (ue) is still negative. m = muscle. In the wound (panel C), TN is
also still absent from the wound epithelium (we). The wound matrix
(wm), however, is strongly TN-positive. Magnification of A, 66 x ; B,
108x; C, 1 0 8 ~ .
Fig. 6. Cryostat sections through normal skin on a n unwounded
limb and wound epithelium from a 24 hr type I wound, both of which
were stained with a polyclonal antibody to TN. The normal skin on
the left was taken from the contralateral limb of the animal providing
the wound epithelium. Note that TN immunoreactivity is greatly reduced in the wound epithelium (we) compared to normal epidermis
h e ) . g = glands, m = muscle. Magnification, 125 x .
100
80
60
40
20
0
COLL
TN
2001250
100
TN
250
MY0
MY0
100
250
Conc. (pg/ml)
Fig. 7. The epidermal response to TN as a migration substrate. Skin
explants were cultured for 16 hr in dishes coated with the indicated
proteins. An ELISA assay of dishes coated with various concentrations of TN indicated that TN binding saturated a t 250 pgiml. The
extent of epidermal migration in each group was determined by
planimetry. N = 5 for myoglobin a t 100 pgiml; N 2 6 for myoglobin
a t 250 pgiml and for both TN groups. The lines on each bar indicate
the S.E.
458
D.J. DONALDSON ET AL.
priller, 1980). Alternatively, it could be that uninjured
epidermal cells normally interact with the anti-adhesive domain of TN, a situation which could promote
structural stability of the epidermis by inhibiting potentially disruptive interactions with other adhesive
proteins. In this case, the loss of TN following wounding would allow epidermal cells to interact with migration-promoting proteins in the basement membrane
and the wound. The loss of TN could be accomplished
by the proteolytic events that are known to occur following wounding. Interestingly, we have found that
certain protease inhibitors block migration (unpublished data). Whether this is related in any way to TN
retention following wounding remains to be established.
When pieces of newt skin were placed in TN-coated
dishes, epidermal migration was quite poor, suggesting
that even if a small amount of TN was present in the
provisional wound matrix, it would not likely be among
the proteins providing a suitable substrate for wound
closure. This conclusion carries a slight caveat, namely, a reminder t h a t by necessity heterologous TN was
used to coat the dishes in these experiments. While this
fact should be kept in mind, it should also be noted that
heterologous fibronectin, fibrinogen, collagen, and vitronectin have all been shown to promote abundant migration of newt epidermal cells in this in vitro system
(Donaldson e t al., 1987). Similarly, Probstmeier et al.
(1990) reported that the intestinal epithelial cell line
HT-29, derived from a human colon adenocarcinoma,
adheres to substratum-immobilized mouse laminin,
various non-human collagens (rat type I, chick type 11,
bovine type 111, and mouse type IV) but not to mouse
JlITN.
The results presented here also show that the loss of
epidermal TN and its widespread expression in mesenchymal tissues of the newt do not depend on the epigenetic mechanisms that regulate appendage regeneration. Simple wounding is sufficient to cause both
events. The function of TN in healing mesenchymal
tissues is a s problematic as in normal epidermis. Since
the cells in these two compartments are so different in
other ways, they may also react differently to TN.
Though it does not appear to mediate migration of epidermal cells, it could do so for mesenchymal cells. Alternatively, i t could modulate the interaction of these
cells with other ECM proteins such as FN (Tan et al.,
1987; Chiquet-Ehrismann et al., 19881, optimizing
them a s migration substrates rather than minimizing
them a s we have suggested for epidermis. Or i t may act
as a mitogen (Chiquet-Ehrismann et al., 1986).Whatever its role in the epidermis or mesenchymal tissues,
the dramatic shift in its expression following wounding
suggests that it exerts a major influence in both places.
ACKNOWLEDGMENTS
This study was supported by NIH grants AR27940,
BRSG RR05423 and DK04256.
LITERATURE CITED
Arsanto, J.-P., M. Diano, Y. Thouveny, J.P. Thiery, and G. Levi 1990
Patterns of tenascin expression during tail regeneration of the
amphibian urodele Pleurodeles waltl. Development, 109:177-188.
Bourdon, M.A., and E. Ruoslahti 1989 Tenascin mediates cell attachment through an RGD-dependent receptor. J. Cell Biol., 108:
1149-1155.
Bourdon, M.A., T.J. Matthews, S.V. Pizzo, and D.D. Bigner 1985 Immunochemical and biochemical characterization of a glioma-associated extracellular matrix glycoprotein. J. Cell Biochem., 28:
183-195.
Chiquet, M., and D.M. Fambrough 1984a Chick myotendinous antigen. I. A monoclonal antibody as a marker for tendon and muscle
morphogenesis. J. Cell Biol., 98t1926-1936.
Chiquet, M., and D.M. Fambrough 1984b Chick myotendinous antigen. 11. A novel extracellular glycoprotein complex consisting of
large disulfide-linked subunits. J. Cell Biol., 98t1937-1946.
Chiquet-Ehrismnnn, R., E.J. Mackie, C.A. Pearson, and T. Sakakura
1986 Tenascin: An extracellular matrix protein involved in tissue
interactions during fetal development and oncogenesis. Cell, 47;
131-139.
Chiquet-Ehrismann, R., P. Kalla, C.A. Pearson, K. Beck, and M. Chiquet 1988 Tenascin interferes with fibronectin action. Cell, 53:
383-390.
Clark, R.A.F., J.M. Lanigan, P. DellaPelle, E. Manseau, H.F. Dvorak,
and R.B. Colvin 1982 Fibronectin and fibrin provide a provisional
matrix for epidermal cell migration during wound reepithelialization. J. Invest. Dermatol., 79t264-269.
Donaldson, D.J., and J.T. Mahan 1983 Fibrinogen and fibronectin as
substrates for epidermal cell migration during wound closure. J.
Cell Sci., 62.117-127.
Donaldson, D.J., and J.T. Mahan 1984 Epidermal cell migration on
laminin-coated substrates: Comparison with other excracellular
matrix and non-matrix proteins. Cell Tissue Res., 235t221-224.
Donaldson, D.J., J.T. Mahan, and G.N. Smith, J r . 1987 Newt epidermal cell migration in uitro and zn uiuo appears to involve ArgGly-Asp-Ser receptors. J. Cell Sci., 87t525-534.
Donaldson, D.J., J.T. Mahan, and G.N. Smith, J r . 1988 Newt epidermal cell migration over collagen and fibronectin involves different mechanisms. J. Cell Sci., 90t325-333.
Erickson, H.P., and M.A. Bourdon 1989 Tenascin: An extracellular
matrix protein prominent in specialized embryonic tissues and
tumors. Annu. Rev. Cell Biol., 55'-92.
Erickson, H.P., and J.L. Iglesias 1984 A six-armed oligomer isolated
from cell surface fibronectin preparations. Nature, 311t267-269.
Friedlander, D.R., S. Hoffman, and G.M. Edelman 1988 Functional
mapping of cytotactin: Proteolytic fragments active in cell-substrate adhesion. J. Cell Biol., 107t2329-2340.
Grumet, M., S.Hoffman, K.L. Crossin, and G. M. Edelman 1985 Cytotactin, an extracellular matrix protein of neural and nonneural tissues that mediates glia-neuron interaction. Proc. Natl.
Acad. Sci. U.S.A.,82:8075-8079.
Halfter, W., R. Chiquet-Ehrismann, and R.P. Tucker 1989 The effect
of tenascin and embryonic basal lamina on the behavior and morphology of neural crest cells in uitro. Dev. Biol., 132t14-25.
Hoffman, S., K.L. Crossin, and G. Edelman 1988 Molecular forms,
binding functions, and developmental expression patterns of cytotactin and cytotactin-binding proteoglycan, an interactive pair
of extracellular matrix molecules. J. Cell Biol., 106t519-532.
Kruse, J., G. Keilhauer, A. Faissner, R. Timpl, and M. Schachner
1985 The J1 glycoprotein-A novel nervous system cell adhesion
molecule of the L2iHNK-1 family. Nature, 316t146-148.
Lightner, V.A., F. Gumkowski, D.B. Bigner, and H.P. Erickson 1989
Tenascinihexabrachion in human skin: Biochemical identification and localization by light and electron microscopy. J. Cell
Biol., Z08t2483-2493.
Mackie, E.J., R. Chiquet-Ehrismann, C.A. Pearson, Y. Inaguma, K.
Taya, Y. Kawarada, and T. Sakakura 1987 Tenascin is a stromal
marker for epithelial malignancy in the mammary gland. Proc.
Natl. Acad. Sci. U.S.A., 84t4621-4625.
Mackie, E.J., W. Halfter, and D. Liverani 1988 Induction of tenascin
in healing wounds. J. Cell Biol., 107t2757-2767.
Mahan, J.T., and D.J. Donaldson 1986 Events in the movement of
newt epidermal cells across implanted substrates. J. Exp. Zool.,
237:35-44.
Onda, H., D.J. Goldhamer, and R.A. Tassava 1990 An extracellular
matrix molecule of newt and axolotl regenerating limb blastemas
and embryonic limb buds: Immunological relationship of MT1
antigen with tenascin. Development, 108t657-668.
Probstmeier, R., R. Martini, and M. Schachner 1990 Expression of
Jlitenascin in the crypt-villus unit of adult mouse small intestine: Implications for its role in epithelial cell shedding. Development, I09:3 13-321.
Repesh, L.A., and J.C. Oberpriller 1980 Ultrastructural studies on
migrating epidermal cells during the wound healing stage of re-
TENASCIN AND SKIN WOUNDS
generation in the adult newt, Notophthalrnus uiridescens. Am. J.
Anat., 159:187-208.
Spring, J., K. Beck, and R. Chiquet-Ehrismann 1989 Two contrary
functions of tenascin: Dissection of the active sites by recombinant tenascin fragments. Cell, 59,325-334.
459
Tan, S.S.,K.L. Crossin, S. Hoffman, and G . Edelman 1987 Asymmetric expression in somites of cytotactin and its proteoglycan ligand
is correlated with neural crest cell distribution. Proc. Natl. Aca.
Sci. U.S.A., 84t7977-7981.
Документ
Категория
Без категории
Просмотров
0
Размер файла
919 Кб
Теги
adults, tenascin, wounds, localization, newt, viridescens, notophthalmus, skin
1/--страниц
Пожаловаться на содержимое документа