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Regular Articles |
From the Collagen Research Unit, Biocenter and Department of
Medical Biochemistry,*
Department of
Dermatology,
and Department of
Pathology,
University of Oulu, Finland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1(XVIII) chain
variants and against NC1 sequences specific to the long variant and
were used in combination with in situ hybridization to
localize this collagen in a number of human tissues. They were also
used for Western blotting, which resulted in detection of
overlapping high-molecular weight bands above the 200-kd standard in a
kidney extract. Heparin lyase II and heparin lyase III digestions of
kidney and placenta extracts indicated that at least in these
tissues, type XVIII collagen contains heparin sulfate
glycosaminoglycan side chains. Type XVIII collagen was found to be a
ubiquitous basement membrane component, occurring prominently
at vascular and epithelial basement membranes throughout the body.
Comparison of the expression of the NC1-493 and NC1-303 variants
revealed marked differences. The short variant was found in most
conventional basement membranes, including blood vessels and
the various epithelial structures, and around muscular
structures. The long variant was expressed very strongly in
liver, where it was virtually the only variant in the liver
sinusoids, and it occurred only in minor amounts elsewhere.
Thus, the 192 N-terminal residues specific to the long variant
apparently confer some functional property needed above all in the
liver sinusoids, but also at certain other
locations.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1(XVIII) chain form has a motif of 10
cysteine residues homologous to the extracellular part of the frizzled
receptors involved in the Wingless signaling pathway in
Drosophila.10
We have recently reported also the
full-length human type XVIII collagen cDNAs that encode 1516- or
1336-amino acid residue 1(XVIII) chains.11
The two
chains have different signal peptides and variant N-terminal
noncollagenous NC1 domains of 493 (NC1-493) and 303 (NC1-303) amino
acid residues, respectively, but share the last 301 residues of their
NC1 domains, a 688-residue collagenous sequence (COL1 to COL10) with
nine interruptions (NC2 to NC10), and a 312-residue noncollagenous
carboxyl-terminal domain (NC11). The amino acid sequences of the human
and previously characterized mouse 1(XVIII) chains exhibit an
overall identity of 79%, the highest homology being observed in their
last 184 residues, corresponding to a 20-kd proteolytic fragment called
endostatin that is released from the C terminus of the 1(XVIII)
collagen chain and has been shown to inhibit endothelial cell
proliferation, angiogenesis, and tumor growth.12
Our recent Northern analysis of several human adult and fetal tissues shows that human type XVIII collagen is expressed in several tissues in a variant-specific manner.11 The NC1-493 variant mRNAs were mainly seen in liver, whereas other tissues contained only minor or undetectable levels of the mRNAs. Northern hybridizations with a probe specific to the NC1-303 variant virtually lacked the adult liver signal, but they revealed clear hybridization to heart, kidney, placenta, prostate, ovary, skeletal muscle, and small intestine mRNAs and faint hybridization to mRNAs from several other tissues.
Because it is not known which cells are responsible for the synthesis
of type XVIII mRNAs, we performed in situ hybridization
experiments using human fetal and selected adult tissue samples. These
and immunostaining experiments demonstrated that type XVIII collagen is
located ubiquitously in basement membrane (BM) zones, its expression
pattern being almost identical to that of the 1 and 2 chains of
type IV collagen, with only a few exceptions. Marked differences were
found in the location of the variant type XVIII chains.
Furthermore, results were obtained with respect to glycosylation of
this collagen.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Probes corresponding to two regions of the human 1(XVIII) chain
mRNAs were used. HuL8.2-E/P, specific to the long variant, was prepared
by subcloning a 492-bp EcoRI/PstI insert of
HuL8.211
to plasmid Bluescript SK (Stratagene, La Jolla,
CA). The plasmid HuL8.2-E/P was linearized with EcoRI, and
35S-UTP-labeled antisense RNA was obtained with T3 RNA
polymerase and the Riboprobe Combination System in vitro
translation kit (Promega, Madison, WI). Labeled sense RNA was prepared
correspondingly, except that HuL8.2-E/P was linearized with
PstI and T7 RNA polymerase was used.
HL8-E/S, a probe detecting all variants, was prepared by subcloning a 272-bp EcoRI/SacI insert of HL811 to plasmid Bluescript SK (Stratagene). The plasmid HL8-E/S was linearized with EcoRI, and the 35S-UTP-labeled antisense RNA was obtained with T3 RNA polymerase. The sense RNA was obtained by linearizing the plasmid HL8-E/S with SacI and by using the T7 RNA polymerase. The probes in the sense orientation served as controls for nonspecific hybridization.
Tissue Sections and in Situ Hybridization Analysis
Several normal placentas, tissues from two 20-gestational week fetuses with Down's syndrome, and a 19-gestational week fetus with pulmonary adenoid malformation were obtained from legal abortions. Normal skin, skeletal muscle, and liver samples from adult persons were also obtained. Some of the samples were fixed in 10% formalin and embedded in paraffin for routine histology. For in situ hybridization, part of each sample was frozen in liquid nitrogen for cryosections, and another part was fixed in 4% paraformaldehyde and embedded in paraffin.
The in situ hybridizations of paraffin sections were carried out according to Hogan et al13 and Hoeffler et al,14 with minor modifications.15 The only difference compared with our previous procedure15 was that washing in 2x standard saline citrate at 45°C was followed by washing in 0.2x standard saline citrate at 37°C (twice for 15 minutes each), after which the slides were dehydrated in 30, 50, 75, and 96% ethanol with 0.3 mol/L ammonium acetate and air dried. For autoradiography, the slides were dipped into Kodak NTB-3 nuclear track emulsion diluted 1:1 in 1% glycerol at 45°C and exposed for 8 to 12 days at 4°C. They were then developed in Kodak D-19 developer for 5 minutes at room temperature, fixed for 5 minutes, and counterstained with Gill's hematoxylin no. 1 (Sigma Chemical Co., St. Louis, MO) and eosin (Orion, Espoo, Finland). The cryostat sections were treated by the same procedure as previously described.15
Expression of Polypeptide Fragments in E. coli for Antigen Production
A 700-bp cDNA fragment, QH48, corresponding to the common region of the NC1 domain of human type XVIII collagen, was generated by polymerase chain reaction using HL411 as a template and primers H18-HIS-4 and H18-HIS-8 (see below). The fragment was subcloned after KpnI/HindIII digestion into the vector pQE-41 (Qiagen, Inc., Santa Clarita, CA), which can be used to express in-frame dihydrofolate reductase (DHFR) fusion proteins with an N-terminal His-tag (clone QH48) and transformed into the Escherichia coli strain M15. The QH48 fragment was expressed as suggested by Qiagen; the bacterial culture was pelleted and resuspended in 6 mol/L guanidine-HCl, 0.5 mol/L NaCl, 20 mmol/L Tris-HCl, pH 7.9; frozen in -70°C; and lysed at room temperature for 1 hour. The suspension was centrifuged for 20 minutes at 12,000 x g, the supernatant was lightly sonicated, and 5 mmol/L imidazole was added, after which it was applied to a 0.75 x 5 cm ProBond column (Invitrogen, Leek, the Netherlands) pre-equilibrated with 8 mol/L urea, 0.5 mol/L NaCl, and 20 mmol/L Tris-HCl, pH 7.9. The denatured polypeptides bound to the column were allowed to renature by means of a slow stepwise decrease in the concentration of urea for 2 hours in the buffer of 0.5 mol/L NaCl and 20 mmol/L Tris-HCl, pH 7.9. The polypeptides were eluted using a stepwise imidazole gradient from 0 to 1 mol/L in 0.5 mol/L NaCl and 20 mmol/L Tris-HCl, pH 7.9. The eluted fractions were monitored at A280 and verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie staining. The fractions containing the human type XVIII collagen-derived polypeptide were dialyzed against 1x phosphate-buffered saline (PBS) and concentrated by ultrafiltration (molecular weight cut of 10 kd, Millipore, Bedford, MA).
A 430-bp fragment QH67, corresponding to the region specific to the long NC1 domain, was amplified as above, except that the template was a genomic subclone HE1.2 of the human COL18A1 gene (H. Elamaa et al, unpublished data) and the primers were H18-HIS-6 and H18-HIS-7 (see below). This polymerase chain reaction product was subcloned into the vector pQE-41, and the clone QH67 was expressed and purified as for QH48 above.
Antibodies to the fusion proteins QH48 and QH67 were raised by conventional methods. For immunization, 300 µl of purified fusion protein solution was injected into rabbits subcutaneously with complete Freund's adjuvant (Sigma Chemical Co.) followed by booster injections with incomplete Freund's adjuvant (Sigma Chemical Co.) at intervals of 14 days. The sera were tested by Western blotting using crude bacterial cell lysates from the expressed clones, QH48.18 and QH1415, encoding type XVIIII collagen sequences without the DHFR leader sequences (see below). Oligonucleotides used were the following (KpnI and HindIII restriction sites are underlined): H18-HIS-4, 5'-ATAAGCTTACGTGGAGACAGAATC-3'; H18-HIS-8, 5'-ATGGTACCCAGCCTCTTCTTCC-3'; H18-HIS-6, 5'-ATAAGCTTAGGGCCCCGTGAGTGG-3'; and H18-HIS-7,5'-ATGGTACCCCGGAATGGTTCCA-3'.
Affinity Purification and Characterization of Human Type XVIII Collagen-Specific Antibodies
Two additional bacterial expression constructs were made to aid affinity purification of the sera. The expression clone QH48.18 was the same as QH48, except that the insert was subcloned into the vector pQE-31 (Qiagen, Inc.), which can be used to express polypeptides with an N-terminal His-tag without the DHFR leader. A slightly longer construct than QH67, a 550-bp fragment QH1415.7, was produced using the primers H18-HIS-14 and H18-HIS-15 (H18-HIS-14, 5'-ATGGTACCCTGGTTCAATAATGAGG-3'; H18-HIS-15, 5'-ATAAGCTTATGAAGATGGTGGTGGC-3'; KpnI and HindIII restriction sites are underlined) and subcloned into the vector pQE-31.
The fragments were expressed and purified as above, except that an additional purification step was included for the QH1415.7 fragment. This was dialyzed in 20 mmol/L piperazine, pH 5.5, after the ProBond column and applied to a HiTrap Q anion-exchange column according to the manufacturer's instructions (Pharmacia, Uppsala, Sweden), and the bound polypeptides were eluted with a stepwise gradient of NaCl from 0 to 1 mol/L in 20 mmol/L piperazine, pH 5.5. The positive fractions of QH48.18 and QH1415.7 after the ProBond and HiTrap Q column, respectively, were pooled and dialyzed in 0.5 mol/L NaCl, 0.2 mol/L NaHCO3, pH 8.6, after which they were coupled to CNBr-Sepharose (Pharmacia) according to the manufacturer's protocol. The identities of the isolated polypeptides were confirmed by sequencing their N-terminal ends using automated Edman degradation with an Applied Biosystems model 477A protein sequencer (Department of Medical Biochemistry, University of Oulu, Finland).
For affinity purification of the type XVIII collagen-specific antibodies, the antisera were diluted 1:1 with PBS, pH 7.4, and applied to the respective columns, which were subsequently washed with 2 mol/L NaCl-PBS, pH 7.4. The bound antibody molecules were eluted with 150 mmol/L glycine-HCl, pH 2.5, and then with 100 mmol/L triethylamine, pH 11.0. The fractions containing protein were detected at A280, immediately neutralized with 2 mol/L Tris-HCl, pH 7.5, pooled, and concentrated (molecular weight cut of 100 kd, Millipore) with 1x PBS.
One-milliliter aliquots of the isopropyl-ß-D-thiogalactopyranoside-induced bacterial cell cultures of DHFR, QH48, H48.18, QH67 and QH1415.7 were centrifuged, suspended in 200 µl of 20 mmol/L sodium phosphate, pH 7.2, and treated for analysis under reduced conditions by 12% SDS-PAGE followed by staining with Coomassie Brilliant Blue and Western blotting onto nitrocellulose filters. The affinity-purified antibodies were used at dilutions of 1 µg/ml for 2 hours at room temperature and detected with a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Bio-Rad, Richmond, CA) and enhanced chemiluminescence detection reagents (Amersham Corp., Buckinghamshire, UK) as recommended. The antibodies were also characterized by using a kidney tissue sample. Thirty micrograms of Human Kidney Protein Medley sample (Clontech, Palo Alto, CA) with 5% 2-mercaptoethanol was boiled for 5 minutes, applied to a reduced 7% SDS-PAGE, and treated for Western blotting using the type XVIII collagen antibodies. The immunosignals were detected using enhanced chemiluminescence as above.
Characterization of the Glycosaminoglycan Side Chains Attached to Type XVIII
Crude proteoglycan fractions were isolated from a normal human 10-gestational week placenta and a kidney obtained after surgical transplantation of a patient with congenital nephrosis, Finnish type, as described.16 Shortly, 0.83 g of placenta and 0.33 g of kidney were homogenized in 10 ml of 4 mol/L guanidine-HCl, 4% 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate (CHAPS, Boehringer Mannheim), 0.1 mol/L sodium acetate, pH 5.8, with proteinase inhibitors (Complete EDTA-free, Boehringer Mannheim) for 24 hours at 4°C. The suspensions were then centrifuged at 12,000 x g for 15 minutes, and the supernatants were dialyzed overnight in 8 mol/L urea, 0.1 mol/L NaCl, and 0.1% CHAPS, pH 7.5. The crude proteoglycan fractions were isolated by anion-exchange chromatography (Q-Sepharose, Pharmacia) in the urea buffer. The samples flowed through were discarded, and all of the materials bound to the column were eluted in a single step using 8 mol/L urea, 1.5 mol/L NaCl, 0.1% CHAPS, pH 7.5; dialyzed against distilled water; and lyophilized. Samples corresponding to 200 and 85 mg of the placenta and kidney starting material, respectively, were dissolved in 100 µl of 60 mmol/L sodium acetate and 50 mmol/L Tris-HCl, pH 8.0 (for chondroitinase ABC), 50 mmol/L sodium phosphate, pH 7.1 (for heparin lyase II), or 50 mmol/L NaCl, 50 mmol/L Tris-HCl, pH 7.3 (for heparin lyase III with collagenase). Twenty-five milliunits of chondroitinase ABC (Boehringer Mannheim) or 4 mIU of heparin lyase II or III (Sigma Chemical Co.) were added to 27-µl samples in the buffers mentioned above and incubated at 37°C for overnight. After chondroitinase ABC and heparine lyase II digestions, 10 µl of 4x sodium dodecyl sulfate sample buffer and 1 µl of ß-mercaptoethanol were added. Bacterial collagenase digestions were performed after heparin lyase III digestions. For that, 1 µl 0.5 mol/L CaCl2 and 10 U of bacterial collagenase (chromatographically purified; Worthington Biochemical Corp., Freehold, NJ) were added in a reaction volume of 40 µl and incubated at 37°C for additional 4 hours, after which sodium dodecylsulfate sample buffer and ß-mercaptoethanol were added as above. Control digestions were treated in the same way but without added enzymes. Each of the digestions was divided into two SDS-PAGE samples for Coomassie Brilliant Blue staining or Western blotting, which were run in a 7% denatured SDS-PAGE gel. Anti-all human (hu) XVIII and anti-long huXVIII antibodies were used as above in enhanced chemiluminescence detection.
Immunofluorescence Staining of Tissues
Tissue sections from the heart, kidney, liver, lung, and pancreas, from an apparently healthy 17-gestational week male fetus and from two 10-gestational week placentas obtained from therapeutic abortions were analyzed. The mature tissues analyzed included samples from normal adult skin, liver, kidney, and striated neck muscle.
Tissue sections for indirect immunofluorescence staining were
immediately frozen in liquid nitrogen and cut into 5-µm cryosections
on SuperFrost Plus glass slides (Menzel Gläser). The sections
were fixed in precooled ethanol for 10 minutes at -20°C, and
unspecific antibody binding was blocked by incubation with 3% bovine
serum albumin in PBS, pH 7.3, or with 5% fat-free milk powder in PBS,
pH 7.3, for 60 minutes at room temperature followed by overnight
incubation at 4°C with a specific antibody. The antibodies used were
those against QH48.18 and QH1415.7 at concentrations of 10 µg/ml or
20 µg/ml in 3% bovine serum albumin/PBS, pH 7.3, respectively; a
monoclonal antibody to type IV collagen diluted 1:100 (DAKO Corp.,
Copenhagen, Denmark) as a marker for BMs; a monoclonal antibody to CD34
diluted 1:25 (Novocastra Laboratories Ltd., Newcastle, UK) as a marker
for endothelial cells; a monoclonal antibody to -smooth muscle actin
(Sigma Chemical Co.) as a marker for smooth muscle cells; and a
monoclonal antibody to vascular endothelial growth factor receptor,
FLT4 diluted 1:200 (a generous gift from Professor Kari Alitalo,
Haartman Institute, University of Helsinki, Finland), as a marker for
lymphatic vessels. After thorough washing with PBS, pH 7.3, a
tetrarhodamine isothiocyanate-conjugated polyclonal swine anti-rabbit
antibody (DAKO Corp.) or rabbit anti-mouse antibody (DAKO Corp.) was
applied, and the samples were incubated for 60 minutes in the dark at
room temperature. A fluorescein isothiocyanate-conjugated goat
anti-mouse antibody (DAKO Corp.) was used for the
double-immunofluorescence stainings. The slides were then mounted with
Glysergel (DAKO Corp.) and examined under an epifluorescence microscope
(Leitz Aristoplan) equipped with filters for tetrarhodamine
isothiocyanate and fluorescein isothiocyanate fluorescence. Control
sections were stained with the secondary antibody alone. The
specificity of the stainings was further demonstrated by blocking the
immunostainings by incubating the affinity-purified type XVIII
antibodies overnight at 4°C with solutions of the corresponding human
type XVIII collagen-derived polypeptides, ie, QH48.18 and QH1415.7, and
using this mixture for additional immunostainings. For better
histological analysis, frozen sections from all the tissues examined
were stained with hematoxylin and eosin by routine methods.
To reduce the possibility of the epitopes recognized by type XVIII collagen-specific antibodies being masked in tissues, some of the sections were pretreated with 6 mol/L urea, 0.1 mol/L glycine, pH 3.5, for 1 hour at 4°C, with 10 U/ml bacterial collagenase (Worthington Biochemical Corp.) for 2 hours at 37°C, with 5% hyaluronidase (Sigma Chemical Co.) for 15 minutes at 37°C, with 0.2 U chondroitinase ABC (Boehringer Mannheim) at 37°C overnight, with 0.2 U N-glycosidase (Boehringer Mannheim) at 37°C for overnight, or with 0.7 U heparin lyase II and III (Sigma Chemical Co.) at 37°C for overnight before blocking by incubation with 3% bovine serum albumin in PBS.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The construct QH48, corresponding to all type XVIII variants, and
QH67, corresponding to the long variant, were expressed as DHFR fusion
proteins in E. coli (Figure 1)
, and the purified recombinant proteins
were used to immunize rabbits. The ensuing sera were affinity purified
using type XVIII polypeptide fragments expressed without the DHFR
leader sequences to isolate type XVIII-specific antibodies designated
as anti-all huXVIII and anti-long huXVIII antibodies (see Materials and
Methods).
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. In the case of the anti-long
huXVIII antibody, no immunosignal was visible using the DHFR sample,
whereas 44- and 33-kd bands were detected with the QH67 and QH1415.7
constructs, respectively (the latter lacking the DHFR sequence) (Figure 2B)
. Thus, the anti-long huXVIII antibody detected polypeptide
sequences derived from the long type XVIII variants. A minor 32-kd band
was also detected in the QH67 sample, which was most probably a
degradation product from QH67, because it was not detected in the DHFR
or QH1415.7 samples.
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. The anti-all huXVIII
antibody resulted in a broad, smear-like band above the highest 200-kd
molecular weight standard, as is typical of proteoglycans containing
long GAG side chains of different lengths.
Interestingly, the Western analysis with the anti-long huXVIII resulted
in a similar smear-like signal, but with less intensity (Figure 2C)
.
Moreover, the signal with the anti-long huXVIII antibody overlapped the
one obtained with the anti-all huXVIII antibody, but consisted only of
the upper part of the broad smear detected with the latter, a finding
consistent with the expected polypeptide lengths of the long and short
variants. Also, the intensities of the bands were in agreement with the
results obtained by immunohistochemistry (see below, Figure 6
), in
which the anti-long huXVIII gave far weaker immunosignals than the
anti-all huXVIII. The specificity of the immunostainings was further
demonstrated by incubating the affinity-purified type XVIII antibodies
with molar excess solution of the corresponding human type XVIII
collagen-derived polypeptides and using these mixtures for additional
stainings. Immunostaining of tissue sections with these mixtures did
not show any immunoreactivity (data not shown). These results suggest
that the two antibodies can be used to detect human type XVIII collagen
in a specific manner.
|
. In contrast to the commercial normal
kidney sample, we were not able to detect type XVIII in a nondigested
proteoglycan fraction isolated from a patient (Figure 3
, lanes 3 and
5), probably because of the different nature of the samples. However,
after heparin lyase II and III digestions, strong signals of 180 kd
were detected (Figure 3
, lanes 4 and 9). The normal placenta sample
gave identical results but with very weak signal intensities (data not
shown). Thus, type XVIII contains heparan sulfate side chains at least
in some tissues. Chondroitinase ABC did not have any effect compared
with the nondigested samples (data not shown). The collagenous nature
of the 180-kd band was verified by demonstration that after heparin
lyase III digestion, the band was sensitive to bacterial collagenase
(Figure 3
, lane 10). The anti-long huXVIII antibody was negative in all
samples studied (data not shown), whereas in the commercial sample it
gave a weak smear (Figure 2C)
. This difference was likely due to the
fact that the tissue section from the same patient sample almost
totally lacked glomeruli, which are virtually the only structures in
kidney positive for the anti-long huXVIII antibody in
immunofluorescence stainings.
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In situ hybridization was performed on paraffin and
cryosections from five normal placentas and on sections representing
eight types of tissue from two fetuses with Down's syndrome and a
fetus with adenoid malformation of the lung (Table 1)
. The sections included samples from
the heart, liver, kidney, pancreas, skin, lung, thymus, and bone. Adult
human skin and liver sections were also analyzed by in situ
hybridization (Table 1)
. Two antisense RNA probes, HL8E/S, covering
some of the sequences encoding the NC1 domain region common to all type
XVIII collagen chains, and HuL8.2E/P, covering sequences specific to
the long cDNA variant were used for the hybridizations (Figure 1)
. The
same probes in the reverse orientation (sense RNA) served as negative
controls for nonspecific hybridization. Only an evenly distributed
background of autoradiography grains was seen with the sense probes.
The results with respect to specific hybridization were generally the
same in the cryosections and paraffin sections.
|
. Double-immunofluorescence staining
and stainings of adjacent sections using an antibody to type IV
collagen were used to verify the location of the type XVIII collagen
signal in relation to the BMs in the tissue samples. The same approach
was also adopted with CD34, -smooth muscle actin, and FLT4
antibodies to aid in the identification of endothelial cells, smooth
muscle structures, and small lymphatic vessels, respectively.
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The human placenta gave an intense signal for collagen XVIII mRNAs
in the cells of the double-layered trophoblastic epithelium of the
villi and in the unorganized cytotrophoblasts of the trophoblastic
columns, including multinucleated syncytial giant cells, with the probe
HL8E/S, encoding sequences common to all 1(XVIII) chains (Figure 3
,
A and B). The signals in the endothelial cells of the villi were
consistent, although not as strong as in the epithelial cells, whereas
labeling of the villous myofibroblasts was weak (Figure 3
, A and B).
The stromal cells of the gestational endometrium seemed to express the
mRNAs faintly, whereas the signal in the epithelial cells of the
endometrial glands and in the large decidual cells of the decidual
membrane was considerably stronger (not shown). The pericystic cells
and the endothelial cells of the placental spiral arteries were
also clearly positive with the probe HL8E/S (not shown).
The BM zone under the double-layered trophoblastic epithelium and the
blood vessels of the stromal villi showed positive staining with the
anti-all huXVIII antibody (Figure 4
, C
and E), and the fibrovascular stroma of the villi was also positive,
particularly in the case of fibrotic villi. There was also staining for
type XVIII collagen around individual cytotrophoblasts of the
trophoblastic columns and around some stromal myofibroblasts of the
villi (Figure 4C)
. Immunofluorescence analysis of the
decidual membrane of the placenta using the anti-all huXVIII
antibody showed a strong positive staining in the maternal blood
vessels and a faint staining lining the epithelial cells in the
endometrial glands (not shown).
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Interestingly, the hepatocytes of both the fetal and adult liver
expressed type XVIII collagen mRNAs. In situ hybridization
studies showed clear hybridization signals in hepatocytes (Figure 5
, A and B). The endothelial cells and
the epithelial cells of the bile ducts in the portal areas were also
positive, whereas the erythropoietic cells in the fetal liver were
negative.
|
. In the adult human liver
the anti-all huXVIII antibody gave a clear, linear staining along the
sinusoids (Figure 5E)
. The walls of the arteries of the portal tracts
were very strongly stained, as was the BM zone under the epithelium of
the bile duct (Figure 5E)
. The capillaries in the portal areas also
showed clear immunoreactivity (Figure 5E)
. Kidney
The epithelial cells of the lower part of the nephron
in the developing fetal kidney, especially the collecting
ducts, were clearly labeled for type XVIII collagen mRNAs
using probe HL8E/S (Figure 6
, A and B). A
positive hybridization signal was seen in the unorganized cells of the
metanephric blastema and the cells of the primitive glomeruli (not
shown) and also in the cells of the more mature glomeruli (Figure 6A)
.
The cells of Bowman's capsule were likewise positive (Figure 6A)
,
whereas the epithelial cells of the proximal tubules were negative (not
shown). The vascular structures of the kidney were also strongly
labeled for type XVIII collagen mRNA (not shown).
Immunofluorescence staining of the fetal kidney with the anti-all
huXVIII antibody showed clear staining in the glomerular BMs and in the
BM zones of most of the tubules and of Bowman's capsule (Figure 6C)
.
The metanephric blastemal cells of the subcapsular fetal cortex were
negative to immunofluorescence staining, whereas there was linear
staining in the developing Bowman's capsule around them (not shown).
Some immature glomeruli and the more mature glomeruli toward the
juxtamedullar region demonstrated clear positivity in the mesangium and
in the BMs of the capillaries once the latter had formed (not shown).
The BM zones of Bowman's capsules were always stained. The anti-all
huXVIII antibody showed strong positive staining in the BM zones of the
tubules in the adult human kidney (Figure 6E)
. Bowman's capsule and
the glomerular mesangium were clearly stained, whereas the glomerular
BM staining was considerably weaker than that in the fetal glomeruli
(Figure 6E)
. All of the vascular structures in the fetal and adult
kidney samples were linearly stained with the anti-all huXVIII antibody
(not shown). Treating the kidney tissue with heparin lyase II or III
before incubation with the primary antibody improved the glomerular
staining, whereas treating the tissue samples with several other
enzymes or buffers (see Materials and Methods) did not have any effect
(not shown). This coincides well with the susceptibility of the kidney
extracts for treatment with heparin lyase II and III (Figure 3)
.
Muscular tissues
In situ hybridizations of fetal heart revealed clear
signals both in the endothelial cells and in the cardiac muscle cells
with the probe detecting both type XVIII mRNA variants (Figure 7
, A and B). Immunofluorescence staining
with the anti-all huXVIII antibody showed staining around individual
heart muscle cells and around numerous small capillaries (Figure 7C)
.
This reaction was very similar to that for type IV collagen, except
that the staining in the capillaries was not as intense (not shown).
The larger blood vessels were more strongly stained for type XVIII than
for type IV (not shown).
|
, D and E).
There was also strong staining in the blood vessels between the muscle
bundles (Figure 7
, D and E). Furthermore, strong staining was seen
around the individual Schwann cells and encircling the whole peripheral
nerves located between the muscle cells (Figure 7D)
. The reaction in
some of the small endomycial vessels between individual muscle fibers
was either very weak or nonexistent. Some of these vessels were stained
with a lymphatic vessel-specific antibody, suggesting that the
lymphatic vessels had only low amounts of type XVIII collagen or lacked
it altogether (not shown). Skin
The keratinocytes of the epidermis in adult and fetal skin were found to express type XVIII collagen mRNAs (not shown). The signal was mainly seen in the basal cells, although some positivity could be seen in all cell layers of the epidermis. The epithelial cells of the sweat glands, cells of the hair follicles, and endothelial cells also contained type XVIII mRNAs (not shown).
Strong positive staining with the anti-all huXVIII antibodies was seen
in the dermal-epidermal BM zone of the adult skin, at capillaries, and
around individual smooth muscle cells (Figure 8A)
. The BM region around the sweat
glands and hair follicles (Figure 8A)
and the BM zones of the nerve
fibers showed linear immunofluorescence staining reactions (not shown),
markedly resembling the staining seen with a monoclonal antibody for
type IV collagen (not shown).
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In situ hybridization analysis of the fetal pancreas
revealed a clear signal in the epithelial cells of the developing acini
(Figure 8
, C and D), whereas the ductal epithelial cells were not
convincingly positive (not shown). The endothelial cells of the
capillaries also expressed the mRNAs. Strong linear immunofluorescence
was seen in the BM zone under the epithelium of the pancreatic acini
(Figure 8E)
and in the BM region of the pancreatic ducts. The signal
around the small capillaries situated in the fibrous stroma and between
the developing acinar structures was faint or nonexistent, whereas the
staining around larger vessels was considerably stronger with the
anti-all XVIII antibody (Figure 8E)
.
Lung
Immunostaining of fetal lung tissue with anti-all huXVIII
antibodies showed linearly stained BM zones around the developing
alveolar structures (Figure 8F)
. The BM regions of the bronchi in the
fibrotic areas were also linearly stained, often with a double contour
representing the BM zone under the bronchial epithelium and staining
around the smooth muscle cells of the bronchial wall (Figure 8G)
. The
staining of the numerous small capillaries between the alveoles
varied from very weak to strong in some capillaries and larger vessels
(Figure 8
, F and G). Despite this clear immunofluorescence signal and
detection of the mRNA in the fetal lung by Northern analysis, repeated
in situ hybridization experiments did not reveal a
convincing signal. This may be due to the low amount of the mRNA in the
tissue.
Differential Expression of Short and Long Variants of 1(XVIII)
Collagen Chains
Marked differences were seen in the occurrence of the
NC1-303 and NC1-493 variants of the 1(XVIII) chain. The NC1-303
variant is clearly the main one in most locations, whereas the NC1-493
variant has a more restricted distribution, the strongest stainings for
it being found in the liver sinusoids (Figure 5
, D and F). The
immunostaining together with the Northern and in situ data
suggest that the sinusoidal staining is largely derived from the
NC1-493 variant. In skin, the BM zones of the epidermal-dermal junction
and the hair follicles were stained for the anti-long huXVIII antibody
(Figure 8B)
, but not the other structures within the dermis, including
the BM zones around the smooth muscle cells, indicating that they
contain only the short variant. Interestingly, the anti-long huXVIII
antibody gave a clear immunofluorescence reaction in placenta only
around the fibroblasts of the villi (Figure 4D)
, whereas the other
stained structures of the placental villi contained the short variant
of the collagen.
One notable finding was the lack of staining for the NC1-493 variant in
capillaries and only weak staining in the walls of large arteries
(Figure 4D
, 5F
, and 8H
), compared with strong signals with the
anti-all huXVIII antibody (Figure 4C
; 5
, A, C, and E; 7
, C to E; and
8
, A and E to G). Only the vascular smooth muscle structures contain
the long variant, given that there is staining in the walls of the
larger vessels in all tissues studied but not in other smooth muscle
structures such as the smooth muscle cells in the dermis (Figure 8B)
or
in the bronchial walls (not shown). The strong staining seen with the
anti-all huXVIII antibody in the epithelial BM zones of the bile ducts,
kidney tubules, pancreatic acini and ducts, lung alveoli, and bronchial
structures and in the BM zones of Bowman's capsule and peripheral
nerves, was lacking with the anti-long huXVIII antibody. The anti-long
huXVIII antibody stained the glomerular BM of the fetal kidney, whereas
the glomerular mesangium of the adult kidney was stained but the
glomerular BM only very weakly so (Figure 6
, D and F). No staining for
the long variant could be found around the cardiac muscle cells of the
heart, but faint staining could be seen at the musculotendinal junction
in the skeletal muscle (not shown).
|
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1(XVIII)
chains in cultured mouse embryonic stem cells.9
The human
type XVIII collagen contains several putative sites for N-linked
glycosylation and GAG side chain attachment, but it has not been known
whether any of these sites are functional. The Western analysis of
human kidney extracts showed a broad band typical of proteoglycans
containing multiple side chains of various sizes, suggesting that some
of these putative sites may be in use. Further analyses by heparin
lyase III digestion of the kidney and placenta extracts indicated that
at least in these tissues, type XVIII collagen contains heparin sulfate
glycosaminoglycan side chains. The size difference between the 200-kd
type XVIII collagen in embryonic stem cells9
and the 180-kd
type XVIII in the kidney analyzed here can be explained with species
differences in other posttranslational modifications or with some
differences in the SDS-PAGE running conditions.
Type XVIII collagen was found in virtually all of the BM zones of the
fetal and adult human tissues studied here. A conspicuous finding in
all tissues was immunostaining of the capillaries as well as the larger
blood vessels, which also showed strong staining in the vessel wall.
Previous studies of type XVIII collagen expression in the mouse
detected this collagen most notably in association with blood vessels,
but also in the BM zone of the skin and faintly in Bowman's capsule in
the kidney.9
We found this collagen to be expressed very
clearly in the human fetal and adult kidney in the Bowman's capsule,
the glomeruli, and the tubules and have recently observed it in this
same location in the mouse kidney using antibodies against mouse type
XVIII collagen-derived fragments (M Rehn et al, unpublished results).
In addition, type XVIII collagen was found adjacent to the
trophoblastic epithelium of the placental villi and in the endometrial
glands of the gestational endometrium; adjacent to the skeletal muscle,
smooth muscle, and heart muscle cells; surrounding the peripheral
nerves; at the epidermal-dermal junction and in the dermal appendices
in the skin; under the epithelium of the pancreatic acini and ducts;
and around the alveoli and bronchi in the lung. In the liver it
appeared in a continuous deposit along the sinusoids and surrounding
the biliary ducts. In situ hybridizations demonstrated that
hepatocytes contain marked amounts of 1(XVIII) collagen chain mRNAs,
suggesting that the strong liver expression is mainly due to synthesis
of type XVIII collagen by hepatocytes. This finding is in striking
contrast with other collagens known to be synthesized in the liver, as
they are produced by nonparenchymal cells such as myofibroblasts and
endothelial cells.17,18
In its prominent occurrence at vascular and epithelial BM zones
throughout the human fetus and in the adult tissues studied here, type
XVIII collagen resembles another widely expressed BM component, type IV
collagen, consisting of 1 and 2 chains.19,20
Thus,
type XVIII collagen appears to be a ubiquitous BM component. A new BM
collagen chain suggested as possibly identical to type XVIII collagen
has recently been identified with a monoclonal antibody, JK-132,
originally produced against human type IV collagen.21,22
Immunohistochemically, this antibody reacted exclusively with the
mesangial matrix of the glomeruli in the normal kidney, and it also
reacted strongly with the expanded glomerular matrix found in
diabetes.23
In view of the extensive expression of type
XVIII collagen in the kidney and the fact that the amino acid sequences
of the peptide fragments recognized by JK-13221
were not
found in the collagen sequences,11
JK-132 appears to
recognize a protein that is distinct from type XVIII collagen.
Comparison of the expression of the NC1-493 and NC1-303 variant chains revealed marked differences. The long variant appeared to be virtually the only one present in liver sinusoids and probably around some myofibroblasts in the placental villi, a clearly detectable variant in the epidermal BM and in the glomerular mesangium and glomerular BM during glomerulogenesis, and a minor variant in vessel walls and skeletal muscle. It could not be detected at all in other locations, although several enzymatic pretreatments were performed to minimize the possibility of the epitopes for the human type XVIII-specific antibodies being masked. Thus, the short variant is the one found in most conventional BMs, including those of blood vessels and the various epithelial structures, and around muscular structures. The 192 N-terminal amino acid residues specific to the long variant apparently confer some functional property needed above all in the liver sinusoids, but also at certain other locations. Interestingly, the finding of the long variant around myofibroblasts in the placental villi could signify that the extensive N terminus has some cell-associated functions. The possible importance of the noncollagenous portions of type XVIII collagen is not restricted to the variant N terminus; however, because since the 20 kd C-terminal fragment of the NC11 domain specifically inhibits endothelial proliferation and potently inhibits angiogenesis and tumor growth.12 This finding fits well with the prominent capillary location of type XVIII collagen, suggesting that the C-terminal fragment may represent a source of regulatory activity released by proteolytic cleavage of the intact molecule under conditions of induced angiogenesis. Nevertheless, the prominent synthesis of type XVIII collagen by hepatocytes also suggests the possibility that it may be secreted into the blood and recruited and activated at sites undergoing angiogenesis as needed.
The homologous collagen types XVIII and XV show both similarities and differences in their tissue distribution. Both are found conspicuously around blood vessels and at the epidermal BM. Type XV collagen is not found as widely in subepithelial BMs as type XVIII,7,24 however, and it is virtually lacking in the fetal liver and has a more restricted distribution in the fetal kidney and lung. On the other hand, type XVIII collagen occurs less prominently around skeletal muscle cells and, in contrast to type XV, is virtually absent from the fibrillar collagen matrix. Thus, even though the tissue distributions of the two homologous collagens are partially overlapping, their roles in tissues may be characterized by certain distinctive features.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Supported by grants from the Health Sciences Council of the Academy of Finland, the Sigrid Juselius Foundation, the Finnish Medical Society Duodecim, and FibroGen Inc. (South San Francisco, CA).
Accepted for publication May 6, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1(XVIII) collagen gene to mouse chromosome 10 and human chromosome 21. Genomics 1994, 19:494-499[Medline]
1(XVIII), a collagen chain with frequent interruptions in the collagenous sequence, a distinct tissue distribution, and homology with type XV collagen. Proc Natl Acad Sci USA 1994, 91:4234-42381 chain of mouse type XVIII collagen, partial structure of the corresponding gene, and comparison of the 1(XVIII) chain with its homologue 1(XV) collagen chain. J Biol Chem 1994, 269:13929-139351(XV), characterized by extensive interruptions in the triple-helical region. Proc Natl Acad Sci USA 1992, 89:10144-10148This article has been cited by other articles:
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