Expression of ␤ig-h3 by Human Bronchial Smooth Muscle Cells
Localization to the Extracellular Matrix and Nucleus
Paul C. Billings, David J. Herrick, Pamela S. Howard, Umberto Kucich, Beatrice N. Engelsberg,
and Joel Rosenbloom
Department of Anatomy and Histology, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
Bronchial smooth muscle cells play a central role in normal lung physiology by controlling airway tone. In
addition, airway smooth muscle hyperplasia and hypertrophy are important factors in the pathophysiology
of asthma. In this study, expression of ␤ig-h3, a recently identified component of the extracellular matrix
(ECM), was investigated in primary human bronchial smooth muscle (HBSM) cells. Northern blot analysis demonstrated that treatment of cultured HBSM cells with transforming growth factor-␤1 resulted in a
4- to 5-fold increase in the steady-state level of ␤ig-h3 messenger RNA. Western blot analysis of secreted
proteins using monospecific antibodies generated against peptide sequences found in the N- and C-terminal regions of the protein identified several isoforms having apparent mass of 70–74 kD. Immunohistochemical analysis of human lung localized ␤ig-h3 to the vascular and airway ECM, and particularly to
the septal tips of alveolar ducts and alveoli, suggesting that it may have a morphogenetic role. Analysis of
cultured HBSM cells identified ␤ig-h3 in both the ECM as well as the cytoplasm, and surprisingly also
in the nucleus. These results demonstrate that ␤ig-h3 is produced by resident lung cells, is a component of
lung ECM, and may play an important role in lung structure and function. The presence of this protein in
nuclei suggests that it may have regulatory functions in addition to its role as a structural component of
lung ECM. Billings, P. C., D. J. Herrick, P. S. Howard, U. Kucich, B. N. Engelsberg, and J. Rosenbloom. 2000. Expression of ␤ig-h3 by human bronchial smooth muscle cells: localization to the extracellular matrix and nucleus. Am. J. Respir. Cell Mol. Biol. 22:352–359.
Lung development and function are dependent on the
proper interaction of resident cells with each other and
with specific extracellular matrix (ECM) components (1–
4). In the bronchus, smooth muscle cells reside in a network of fibrous connective tissue below the respiratory epithelium and play a key role in regulating airway tone.
Further, increased proliferation and other alterations in
these cells have been implicated in the pathophysiology
of asthma (5–8). To better understand smooth muscle cell
physiology and its role in specific pathologic conditions, it
is necessary to define the ECM components produced by
(Received in original form March 17, 1999 and in revised form September 8,
1999)
Address correspondence to: Paul C. Billings, Dept. of Anatomy and Histology, University of Pennsylvania School of Dental Medicine, 4010 Locust St., Philadelphia, PA 19104–6002. E-mail: [email protected].
upenn.edu
Abbreviations: antibody, Ab; bovine serum albumin, BSA; complementary DNA, cDNA; dithiothreitol, DTT; extracellular matrix, ECM; ethylenediaminetetraacetic acid, EDTA; fetal bovine serum, FBS; human
bronchial smooth muscle, HBSM; horseradish peroxidase, HRP; keyhole
limpet hemocyanin, KLH; messenger RNA, mRNA; nicotinamide adenine dinucleotide phosphate, NADPH; optimal cutting temperature,
OCT; phosphate-buffered saline, PBS; PBS containing 0.1% Tween 20,
PBST; transforming growth factor, TGF.
Am. J. Respir. Cell Mol. Biol. Vol. 22, pp. 352–359, 2000
Internet address: www.atsjournals.org
these cells, establish their anatomic distribution, and determine the interaction of smooth muscle and other resident lung cells with these proteins. The goal of the current
study was to examine the expression and deposition of
␤ig-h3, a newly described ECM protein, by bronchial
smooth muscle cells.
The ECM is composed of a wide array of macromolecules, including collagens, laminins, elastin, and proteoglycans (reviewed in 9). The components of the ECM interact
with each other to form a highly organized framework that
provides a support structure for resident stromal and parenchymal cells, and maintains tissue architecture. The
interaction of resident cells with the ECM is crucial for
normal development and changes in these interactions
likely play important roles in many pathologic conditions.
Whereas the major components of the ECM have been
identified and extensively characterized, considerably less
is known about factors that interconnect these components
with one another and resident cells in different tissues.
␤ig-h3 was first observed in reductive saline extracts of
fetal bovine nuchal ligament as a protein that migrated
with an apparent molecular weight of ⵑ 70 kD (10). The
protein was subsequently cloned by differential screening
of a complementary DNA (cDNA) library prepared from
A549 human lung cells treated with transforming growth
factor-beta 1 (TGF-␤1) (11). Recently, highly homologous
Billings, Herrick, Howard, et al.: ␤ig-h3 Expression in Lung Cells
cDNAs encoding mouse, chick, pig, and rabbit ␤ig-h3 have
been cloned (12–15). Other proteins having more limited
homology to ␤ig-h3 include periostin (16), Drosophila fasciclin I (17), and several bacterial proteins (18). The nascent
protein contains a secretory signal sequence and is composed largely of four homologous internal domains, the last
one of which contains an arginine–glycine–aspartic acid sequence that may act as a cell attachment/integrin binding
site (11, 12). Whereas the physiologic function of ␤ig-h3
has not been elucidated, the porcine protein has been
shown to bind collagens I, II, and IV (13), and ␤ig-h3 has
been hypothesized to serve a linking function, interconnecting different matrix components with each other and
resident cells (12, 13, 19, 20).
Northern blot analysis demonstrated that ␤ig-h3 was
expressed in a variety of human and mouse tissues with
the highest concentrations found in uterine tissue, and
readily detectable messenger RNA (mRNA) levels were
found in heart, breast, prostate, skeletal muscle, testes,
thyroid, kidney, liver, and stomach tissues (12). Expression was absent in brain, spleen, and parathyroid tissues.
At the protein level, the distribution of ␤ig-h3 has been examined in fetal bovine tissues (19). This analysis showed
that ␤ig-h3 was associated with collagen fibers in developing nuchal ligament, aorta, lung, and mature cornea. Immunoreactive material was also present in reticular fibers
in fetal spleen as well as capsule and tubule basement
membranes in developing kidney. From this study, it was
concluded that the staining pattern closely resembled that
of type VI collagen microfibrils.
Interestingly, missense mutations in this protein at positions 124 (Arg 124 → Cys or His) and 555 (Arg 555 → Gln
or Trp) have been identified as causative mutations in hereditary corneal dystrophies (21). Mutations at these sites
are thought to cause the protein to denature, forming
“amyloidogenic intermediates” that subsequently precipitate in the cornea (22). This condition results in a progressive loss of vision, which can ultimately lead to blindness.
These findings suggest that ␤ig-h3 is important in eye
physiology and that specific changes in protein primary sequence alter its functional role in the corneal ECM. However, no abnormal findings relative to the lung have been
reported in patients with corneal dystrophies.
Whereas ␤ig-h3 has been shown to be present in bovine
tissues (19) and human cornea (22), very little information
is available regarding the distribution of the protein in
other human tissues. The goal of the present study was to
assess ␤ig-h3 expression in the lung with emphasis on airway smooth muscle cells. We show that ␤ig-h3 is present
extensively in lung tissue, localizing to alveoli, alveolar
ducts, and bronchioles in close juxtaposition to bronchial
smooth muscle. Cultured bronchial smooth muscle cells
produce readily detectable amounts of ␤ig-h3 that is deposited in the ECM, and that surprisingly is also found in
the nuclei of these cells.
Materials and Methods
Chemicals and Reagents
Goat-antirabbit-horseradish peroxidase (HRP) and goatantirabbit–rhodamine isothiocyanate conjugates were pur-
353
chased from Sigma (St. Louis, MO). Recombinant human
TGF-␤1 was generously supplied by Genentech (San Francisco, CA). Gradient polyacrylamide minigels were purchased from Novex (San Diego, CA).
Cell Culture
The human bronchial smooth muscle (HBSM) cell strains
used in this study were derived from two healthy male donors, 37 and 16 yr of age (Clonetics, San Diego, CA). No
significant differences were observed between the two cell
strains with respect to their ␤ig-h3 production. The cells
were grown in Smooth Muscle Basal Medium supplemented
with 5% fetal bovine serum (FBS), insulin (5 ␮g/ml), human
recombinant epidermal growth factor (10 ng/ml), human
recombinant fibroblast growth factor (2 ng/ml), gentamicin (50 ␮g/ml), and amphotericin-b (50 ng/ml) at 37⬚C, 5%
CO2. A549 human lung carcinoma cells were grown in RPMI
1640 containing 5% FBS.
Purification of Nuclei
Cultured HBSM cells were trypsinized, pelleted by centrifugation, and resuspended in ice-cold sucrose buffer I (0.32 M
sucrose, 3 mM CaCl2, 2 mM Mg-acetate, 0.1 mM ethylenediaminetetraacetic acid [EDTA], 10 mM Tris [pH 8], 1 mM
dithiothreitol [DTT] and 0.1% Triton X-100). The cells
were lysed with a Dounce homogenizer (Fisher Scientific,
Pittsburgh, PA), and cell breakage was periodically assessed
by microscopic examination of the homogenate. When lysis
was complete, the homogenate was mixed with sucrose
buffer II (2.2 M sucrose, 5 mM Mg-acetate, 0.1 mM EDTA,
10 mM Tris [pH 8], and 1 mM DTT) and layered over a 2-M
sucrose cushion. The nuclei were pelleted by centrifugation
(30,000 ⫻ g, 45 min, at 4⬚C) and resuspended in glycerol
storage buffer (40% glycerol, 5 mM MgCl2, and 0.1 mM
EDTA). For analysis, freshly purified nuclei were spread on
glass slides, fixed for 10 min with phosphate-buffered saline
(PBS) containing 1.5% formalin, and subsequently incubated with anti-␤ig-h3 antibodies (see subsequent section),
or propidium iodide, which specifically stains DNA.
Marker Enzymes
Cytochrome C reductase activity (endoplasmic reticulum/
microsomal marker enzyme) was determined in 25 mM
Tris (pH 7.5), 300 mM CaCl, containing 50 ␮M 2,6-dichloroindophenol, and 50 ␮M nicotinamide adenine dinucleotide phosphate (NADPH) (23). The oxidation of
NADPH was monitored at 340 nm (E340 ⫽ 6.22 mM⫺1 ⫻
cm⫺1 [24]).
Galactosyltransferase activity (Golgi marker enzyme)
was determined using the modified procedure of RensDomiano and Roth (25). Briefly, reactions contained extract (20–50 ␮g protein) in 25 mM N-2-hydroxyethylpiperazine-N⬘-ethane sulfonic acid (Hepes) (pH 7.0), 1 mM
DTT, 0.5% Triton X-100, 40 mM MnCl2, 2 mM adenosine
triphosphate, 20 mM N-acetylglucosamine, and 2 mM uridine diphosphate-[14C]galactose ([New England Nuclear,
Boston, MA]; specific activity 300 mCi/mmol) in a total volume of 150 ␮l. The reactions were incubated for 1 h at 37⬚C
and terminated by the addition of 50 ␮l of 0.2 M EDTA and
passed over 0.5 ml of Dowex 1 resin (chloride form) (Sigma).
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 22 2000
The radiolabeled product [14C]lactosamine was eluted with
1 ml of water and counted in a liquid scintillation counter.
Production and Affinity Purification of Antibodies
and Western Blot Analysis
Three synthetic peptides IGTNRKYFTNCKQWYQRKIC
(residues 55–74, antibody [Ab] 1186), TQLYTDRTEKLRPEMEG-C (residues 118–134 of human ␤ig-h3, Ab
1073), and ALPPRERSRLL-C (residues 549–559, Ab
1077) were synthesized and purified by high pressure liquid chromatography by BioSynthesis (Lewisville, TX).
The second and third peptides were synthesized with an
additional cysteine residue at the carboxy terminus to facilitate their coupling to maleimide activated keyhole limpet hemocyanin (KLH) (Pierce, Rockford, IL) at a substitution ratio of 1 mg peptide/mg KLH. Antibodies were
prepared in rabbits by Cocalico Biologicals (Reamstown,
PA) using the following immunization schedule: an initial
injection of 250 ␮g, followed 3 wk later by three biweekly
injections of 100 ␮g. For affinity purification of antibodies,
individual peptides were immobilized on Sulfolink coupling gel (Pierce). The immunoglobulin G fraction of the
antiserum was passed over the column, which was washed
with PBS (ⵑ 10 column vol; 50 ml) until protein free. Bound
antibodies were eluted with 100 mM glycine (pH 2.5), immediately neutralized with Tris base, and diluted to a concentration of 0.4 mg/ml before storage at ⫺70⬚C (26).
Proteins were resolved on 8 to 16% polyacrylamide
gradient gels under reducing conditions and transferred to
nitrocellulose membranes. After transfer, the membranes
were blocked in PBS containing 0.1% Tween 20 (PBST)
containing 5% nonfat dry milk and subsequently incubated with anti-␤ig-h3 antibodies diluted 1:1,000 in PBST
and 0.1% bovine serum albumin (BSA). Bound antibody
was detected with goat-antirabbit-HRP conjugate, using
chloronaphthol as substrate.
Immunohistochemistry
Human lung tissue was fixed in 1% paraformaldehyde for
2 to 3 h at room temperature, followed by 0.5 M sucrose
infusion, and embedded in OCT imbedding medium (Miles,
Elkhart, IN) freezing compound. Frozen sections were cut
and placed on glass slides. The tissue was treated with 6 M
guanidine-HCl and 100 mM iodoacetamide followed by
treatment with H2O2 to reduce endogenous peroxidase activity (10). The tissue was incubated with primary antibody
overnight, washed and incubated with biotinylated antirabbit IgG followed by streptavidin/HRP conjugate (Amersham, Piscataway, NJ), and developed with diaminobenzidine (Sigma) as substrate.
Cells (2 ⫻ 105) were seeded in sterile eight well Lab
Tek chamber slides (Nunc, Naperville, IL) and grown to
confluence. The cells were washed twice with PBS, fixed
with PBS containing 1.5% formalin for 10 min at 20⬚C,
washed with PBS, and permeabilized with PBS containing
0.1% Triton X-100 and 1% BSA. Next, cells were incubated with primary antibody (diluted 1:400 in PBST, 1%
BSA) at 4⬚C overnight, washed three times with PBST,
and subsequently incubated with a rhodamine-conjugated
goat-antirabbit IgG (diluted 1:500 in PBST, 0.1% BSA) for
1 h at 20⬚C. The slides were washed, coverslipped, and ex-
amined with a Zeiss fluorescent microscope (Carl Zeiss, Inc.,
Thronwood, NY) equipped with epifluorescence optics. Control slides were treated in an identical manner, but with the
addition of 10 ␮g/ml peptide to block the primary antibody.
Northern Blots
Total cytoplasmic RNA was extracted with guanidinium
thiocyanate/phenol chloroform extraction as described
previously (27). RNA was size fractionated on 1% agarose-formaldehyde gels, transferred to a Zeta-Probe membrane (Bio-Rad, Hercules, CA), and hybridized with a 2.1kb ␤ig-h3 cDNA labeled with [32P] using a Ready-To-Go
DNA labeling kit (Pharmacia Biotech, Piscataway, NJ).
RNA loading and transfer were evaluated by probing with
a 1.4-kb glyceraldehyde phosphate dehydrogenase cDNA
probe (28). Equivalent loading and transfer were also verified by quantitative image analysis of ethidium bromide
staining of ribosomal RNA in the blots themselves. The
Figure 1. Immunolocalization of ␤ig-h3 in lung tissue. Prefixed,
human lung tissue was embedded in OCT freezing medium. Frozen sections were cut, treated with guanidine hydrochloride to
unmask ␤ig-h3 epitopes, and incubated with anti-␤ig-h3 Ab 1077.
(A) Note prominent staining of septal tips in alveolar ducts and
developing alveoli (open arrowheads). (B) Note staining of matrix in alveolar walls (double arrowheads) and intense staining of
matrix within wall of a bronchiole (solid arrowhead) and alveoli.
Besides matrix staining within the alveolar walls, there is also
more localized staining (arrows), suggesting the possibility of staining of cell nuclei.
Billings, Herrick, Howard, et al.: ␤ig-h3 Expression in Lung Cells
355
phosphorimages of the filters were digitized (Storm 840;
Molecular Dynamics, Sunnyvale, CA) and the signal levels
were quantified (Image-Quant V5.1 software; Molecular
Dynamics) to determine the relative amounts of mRNA.
Results
Immunohistochemical Localization of ␤ig-h3
in Human Lung
To determine the anatomic distribution of ␤ig-h3 in human lung, tissue from a 2-yr-old child who had suffered an
accidental death was embedded in OCT medium, and
frozen sections were cut and incubated with Ab 1077.
In formaldehyde-fixed tissue, staining with ␤ig-h3 was
negative, suggesting as in previous studies (19), that the
antibody binding sites may be masked. The fixed tissue
sections were treated with guanidine-HCl followed by
iodoacetamide to unmask reactive epitopes. Whereas the
tissue morphology exhibited less than optimal cellular
structures because of the powerful chaotropic activity, the
treatment resulted in positive staining with Ab 1077. In the
tissue, we observed staining of the ECM of the vasculature
(not shown), alveolar ducts, as well as developing and
formed alveolar walls (Figures 1A and 1B). Of particular
interest was the intense staining localized to the septal tips
of alveolar ducts and developing alveoli. Further, there
was prominent staining of the matrix and associated
smooth muscle in bronchioles (Figure 1B). Hence, ␤ig-h3
is a component of pulmonary ECM where it is present in
close approximation to resident smooth muscle cells and
fibroblasts. These results provide strong evidence that the
observations made with HBSM cells in culture (see subsequent section) are an accurate reflection of the behavior of
these cells in vivo.
Expression of ␤ig-h3 by Cultured HBSM Cells
Expression of ␤ig-h3 was assessed in cultured HBSM cells.
Our initial experiments compared ␤ig-h3 mRNA expression by HBSM cells to that of A549 cells, the epithelial carcinoma cell line from which ␤ig-h3 was originally cloned
(11). Northern blot analysis demonstrated that whereas
both cell types expressed ␤ig-h3, constitutive expression by
HBSM cells was about 8-fold higher than that of A549 cells
and comparable to A549 expression after treatment with
TGF-␤ (Figure 2). Treatment of HBSM cells with 1 ng/ml
TGF-␤1 (shown to be an optimal concentration in preliminary experiments) for 48 h resulted in a 4- to 5-fold inducFigure 2. Northern blot analysis of ␤igh3 expression in lung cells. RNA was
extracted from A549 and HBSM cells
and analyzed on Northern blots hybridized with [32P]␤ig-h3 (upper section). Relative expression of ␤ig-h3 was quantified by PhosphorImager analysis (lower
section). A549 cells were untreated or
treated with TGF-␤1 (1 ng/ml) for 24 h;
HBSM cells were untreated. Note that
the constitutive level of expression by
HBSM cells is similar to TGF-␤1–stimulated A549 cells.
Figure 3. Time course of TGF-␤1 increase of ␤ig-h3 mRNA concentration. (A) Confluent cultures of HBSM cells were incubated
with or without 1 ng/ml of TGF-␤1 for the indicated times. (B)
RNA was isolated and analyzed by Northern blot hybridization
for ␤ig-h3 mRNA concentrations as described in MATERIALS AND
METHODS. The values represent the average of determinations
from duplicate cultures.
tion of ␤ig-h3 RNA (Figure 3). These results demonstrate
that HBSM cells constitutively express ␤ig-h3 at a relatively high level, but that exposure of these cells to TGF-␤1
still results in a significant increased mRNA level.
We next investigated ␤ig-h3 expression at the protein
level. For these studies, monospecific, affinity-purified antibodies were prepared against the N-terminal (Ab 1186
and Ab 1073; residues 55–74 and 118–134, respectively)
and C-terminal (Ab 1077; residues 549–559) regions of the
protein. Western blot analysis of proteins secreted into the
conditioned medium, as well as proteins extracted from
the matrix formed by the cultured cells, demonstrated that
all antibodies reacted specifically with a 70 kD protein (Figure 4; only the results with Ab 1077 are shown). Interestingly, whereas several isoforms were identified in the medium, as has been noted by others (12, 19), the protein
Figure 4. Western blot analysis of medium and ECM proteins. HBSM cells were grown
to confluence and removed
from the dishes with PBS and
10 mM EDTA. The remaining matrix was solubilized with
1% sodium dodecyl sulfate
and analyzed on Western blots
under reducing conditions. Protein was detected using Ab
1077. Numbers on right indicate relative molecular mass in kD. Note the presence of multiple
isoforms of ␤ig-h3 in conditioned medium, whereas a much more
restricted pattern is found in the matrix. Identical results were
obtained with Ab 1073.
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extracted from the matrix appeared to contain only the
largest isoform.
Immunohistochemical Localization of ␤ig-h3 in
Cultures of HBSM Cells
The results described previously established that HBSM
cells express ␤ig-h3. Our next objective was to assess ␤igh3 protein deposition in situ. Close and careful examination of the immunohistochemically stained tissue sections
at high magnification suggested that the antibody may
have been localized to the nuclei of some cells. However,
this conclusion was quite tentative because of the distortion of the tissue caused by the treatment required to elicit
reactivity. To evaluate ␤ig-h3 expression, cells were grown
in eight-well slide chambers, fixed, and subsequently incubated with ␤ig-h3 antibodies. In HSBM cultures treated
with Ab 1077 (prepared against the C-terminal region of
the protein), fine strands of ECM stained positively (Figures 5A and 5B). In addition, fine punctate cytoplasmic
staining partially surrounding the nucleus and possibly associated with the golgi and/or rough endoplasmic reticulum was detected (Figure 5B). In some cells, we observed
very fine punctate staining of the nuclei (Figure 5A).
A markedly different staining pattern was detected when
cells were incubated with Ab 1073 (prepared against the
NH2-terminal region). Under these conditions, the nuclear
region exhibited intense positive staining (Figure 6). This
staining pattern was judged to be intranuclear rather than
on the cell surface by focusing above and below the plane of
the cell monolayer. Very fine, faint staining of the ECM was
also observed with this antibody upon overexposure of the
Figure 5. Immunofluorescent
analysis of cultured HBSM
cells. (A, B) Cells were grown
on slide chambers, washed,
fixed, permeabilized, and incubated with Ab 1077. Note
staining of ECM (double arrowheads in A and B), punctate intracellular staining (arrowheads in B), and fine nuclear
localization (arrows in A). (C)
Incubation of Ab 1077 with
peptide 1077 resulted in the
absence of staining.
Figure 6. Immunofluorescent analysis of HBSM cells. (A) Cells
were grown on slide chambers, washed, fixed, permeabilized, and
incubated with Ab 1073. Cell bodies are not visualized, and only
the nuclei stained in a punctate manner (arrowheads). (B) High
magnification of a single cell that has been stained with Ab 1073
with overexposure of the image to demonstrate the weak generalized cytoplasmic staining (twin arrowheads). (C) Incubation of
Ab 1073 with peptide 1073 resulted in the absence of staining.
Billings, Herrick, Howard, et al.: ␤ig-h3 Expression in Lung Cells
357
fluorescent image (Figure 6B). We also observed nuclear
staining when cells were incubated with Ab 1186 (Figure 7),
which recognizes an epitope in the N-terminal region of ␤igh3. For controls, HBSM cells were treated with (1) preimmune serum, (2) Ab 1073, 1077, or 1186, that were preincubated with immunizing peptide (Figures 5C, 6C, and 7A), or
(3) secondary antibody alone. Under these conditions, immunostaining of cells and tissue was consistently negative.
Identification of ␤ig-h3 in the Nucleus of HBSM Cells
Although it is clear that ␤ig-h3 is secreted and becomes
deposited in the ECM (Figures 1 and 5), the results obtained when cells were stained with Ab 1073 and Ab 1186
(Figures 6 and 7) supported the observation with whole
lung sections that ␤ig-h3 is also present in the nucleus of
some cells. To confirm this finding, nuclei were purified
from HBSM cells and examined by immunofluorescence
microscopy. The homogeneity of the nuclear preparations
was established by staining with propidium iodide, which
specifically stains DNA (Figure 8), and found to be pure at
this level of resolution. Furthermore, assessment of subcellular marker enzyme activities (Table 1) demonstrated
that the nuclear preparations were relatively free of contamination by golgi and endoplasmic reticulum and comparable in terms of purity to that obtained by others (29,
30). Incubation of nuclei with Ab 1073 resulted in intense
staining characterized by a stippled, punctate pattern. In
addition, proteins extracted from the nuclei were subjected to Western blot analysis, which demonstrated that
both Ab 1073 and Ab 1077 recognized several isoforms of
␤ig-h3 (Figure 8). These results unequivocally confirm the
presence of this protein in HBSM cell nuclei.
Discussion
In this investigation, we found that HBSM cells constitutively express ␤ig-h3 mRNA, and the level of expression
by these cells was considerably higher (ⵑ 8-fold) than that
of A549 cells (Figure 2). Nevertheless, TGF-␤1 still significantly increased expression by the HBSM cells (Figure 3).
In contrast, we have observed that exposure of HBSM
cells to the proinflammatory cytokine, interleukin 1␤, which
upregulates a number of proinflammatory genes (31), had
no effect on ␤ig-h3 expression levels (data not shown).
Figure 8. Immunologic analysis of purified nuclei of HBSM cells.
Nuclei were purified from cultured HBSM cells as described in
MATERIALS AND METHODS. (A) Propidium iodide staining of purified nuclei preparation. (B) Immunofluorescent analysis of purified
nuclei using Ab 1073. (C) Proteins were extracted from purified
nuclei and subjected to Western blot analysis on 8 to 16% sodium
dodecyl sulfate gels under reducing conditions as described in
MATERIALS AND METHODS using Ab 1077 or Ab 1073 as indicated. Multiple isoforms of ␤ig-h3 were detected with both antibodies. Numbers on right indicate relative molecular mass in kD.
Results from several laboratories have demonstrated
that ␤ig-h3 is a component of the ECM in vivo. Immunohistochemical analysis of bovine tissue previously localized the
protein to the matrix in several tissues, including nuchal ligament, kidney, lung, and eye (19). The protein has also been
localized to the matrix in human cornea (22) and was isolated from a fiber-rich fraction of porcine cartilage (13). In
our study, we used immunofluorescent and Western blot
analyses to demonstrate that ␤ig-h3 was present in matrix
produced by HBSM cells. One predominant form with an
apparent approximate mass of 70 kD was detected in the
matrix, whereas several reactive isoforms, mainly of slightly
lower molecular mass, were detected in the conditioned medium (Figure 4). This suggests that the larger isoform is
preferentially incorporated into the matrix. We have obtained similar results with primary human lung fibroblast
(GM05389) cells (data not shown). In lung tissue, ␤ig-h3 localized to matrix in the alveolar ducts and alveoli as well as
in the bronchioles in close association with smooth muscle.
Therefore, these results confirm that the protein is a compo-
TABLE 1
Activity of marker enzymes in HBSM cells*
Extract
Figure 7. Immunofluorescent analysis of HBSM cells. Cells were
grown on slide chambers, washed, fixed, permeabilized, and incubated with (A) preimmune serum or (B) Ab 1186. Note prominent nuclear staining.
Total cell
Nuclei
Galactosyl Transferase
(␮mol/h/mg protein)
Cytochrome C Reductase
(␮mol/min/mg protein)
3.97 ⫾ 0.4
0.41 ⫾ 0.05
173 ⫾ 15
21.6 ⫾ 7.2
*Galactosyl transferase and cytochrome C reductase activities were assayed
as described in MATERIALS AND METHODS.
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nent of lung matrix and suggest that bronchial smooth muscle cells are one source of ␤ig-h3 in vivo.
Because ␤ig-h3 is secreted and present in ECM, its localization to the nucleus was unexpected. However, the
evidence supporting this conclusion is strong. Ab 1073 and
1186 consistently stained HBSM cell nuclei (Figures 6 and
7) as well as nuclei in other cell types, including human
lung fibroblasts (data not shown). A similar immunostaining pattern was obtained with purified nuclei. Ab 1077 produced a weaker but still positive nuclear staining. In addition, both the N-terminal (1073) and C-terminal (1077)
antibodies reacted with the same nuclear proteins on Western blots, indicating that both regions of the protein are
present in the nuclear and secreted forms of the protein.
These results indicate that the epitope recognized by Ab
1073 (which stains nuclei) is accessible in nuclei, but not in
matrix, whereas the epitope recognized by Ab 1077 (primarily staining matrix) is accessible in matrix, but only
poorly accessible in nuclei. This could reflect the fact that
␤ig-h3 interacts with different ligands, which specifically
bind these sites in these two compartments, thus blocking
antibody binding. Alternatively, the protein may assume
different conformations in these two compartments, altering epitope accessibility and/or antibody recognition.
The presence of this protein in ECM and nuclei raises
several questions regarding protein trafficking. Because
␤ig-h3 contains a signal sequence on the amino terminus
(residues 1–23; see Reference 11), it is predicted that the
protein is targeted for secretion. Indeed, we have detected
immunoreactive material in what appears to be golgi of
HBSM and lung fibroblast cells, consistent with the translocation of this protein to the extracellular space (32, 33). One
possibility to explain nuclear localization is that the protein
is secreted and subsequently taken up by cells, perhaps by
receptor-mediated endocytosis. Alternatively, the protein
may have multiple isoforms arising from alternative splicing
in the 5⬘ region of the mRNA or by post-translational proteolytic processing. These different ␤ig-h3 isoforms could
potentially be targeted to specific compartments; some isoforms are secreted and deposited in the ECM, while other
isoforms that lack the signal peptide are translocated to the
nucleus. We are currently investigating these possibilities.
␤ig-h3 has been shown to enhance attachment and
spreading of dermal fibroblasts, suggesting that it functions as an extracellular attachment protein in skin (20).
Indeed, the protein contains an RGD cell attachment/integrin recognition site in its C-terminal region. Immunofluorescence analysis of bovine tissue showed ␤ig-h3 associated
with collagen fibers (19). In addition, the porcine-derived
␤ig-h3 homologue has been reported to bind types I, II,
and IV collagen (13). These data suggest that ␤ig-h3 may
act as a linker protein interconnecting specific matrix components, such as collagens, with each other and resident
cells. For example, bronchial smooth muscle, or other resident lung cells, could use surface integrins to bind ␤ig-h3,
which in turn, could bind collagen molecules present in the
tissue stroma of the lung. The molecule would then serve a
bridging function, connecting lung smooth muscle, or other
resident cells, with matrix collagens or other ECM compartments. If this is the case, then we speculate that ␤ig-h3
plays an important role in the interaction of lung resident
cells with the ECM and maintenance of lung structure.
It is generally believed that the interactions of lung epithelial, fibroblast, and smooth muscle cells with the ECM
play a critical role in lung development and function. The
localization of ␤ig-h3 to the forming alveolar walls, especially at the tips of newly forming septa, is of particular
interest because elastin is also found at these sites. The localization of a condensed elastic matrix at the apex of
forming alveolar septae suggested that this matrix might
have a critical role in alveolar morphogenesis as opposed
to a purely structural one (34–36).
Alterations in cell/matrix interactions are involved in the
pathogenesis of diseases such as asthma and fibrosis (5–9). In
lung fibrosis, matrix accumulation by lung fibroblasts, and
possibly other cells, proceeds unabated. In this process,
TGF-␤1 has been proposed to act as a master switch, controlling collagen accumulation (37). In this environment, ␤ig-h3
could lead to increased noncovalent “crosslinking” of collagen molecules and other matrix components and also result
in altered cell/matrix interactions. Consequently, changes in
matrix composition, such as increased collagen/␤ig-h3 deposition in lung alveoli and bronchioles, could potentiate the fibrotic process by diminishing lung elasticity and function.
Our results demonstrate that ␤ig-h3 is expressed by bronchial
smooth muscle cells and once secreted, some of the protein
becomes deposited in the ECM. In addition, this protein is
present in nuclei, indicating that it may have other, as yet, unknown functions in addition to its role as a structural ECM
component and potential cell attachment factor.
Acknowledgments: The writers thank Dr. Linda Gonzales for providing them
with human lung tissue. The analytical assistance of Drs. Arthur Cohen and Bill
Abrams is acknowledged and appreciated. This research was supported by NIH
grants HL56401 and DK48215.
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