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*
Department of Pediatrics, Kinki University School of Medicine,
Osaka-Sayama, Japan
Department of Pathology, Institute of Basic Medical Science, University of
Tsukuba, Tsukuba, Japan
Second Department of Internal Medicine, Tokyo Medical and Dental
University, Tokyo, Japan
Department of Medicine, College of Physicians and Surgeons, Columbia
University, New York, New York
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Department of Medicine, Vanderbilt University School of Medicine,
Nashville, Tennessee.
Correspondence to Dr. Kazuo Yoshioka, Department of Pediatrics, Kinki University School of Medicine, 377-2, Ohno-higashi, Osaka-Sayama 589-8511, Japan. Phone: 011-81-723-66-0221; Fax: 011-81-723-68-1566; E-mail: kzyoshio{at}med.kindai.ac.jp
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(TGF-), hepatocyte growth factor (HGF), glial cell line-derived
neutrophic growth factor (GDNF), insulin-like growth factor-1 (IGF-1), and
IGF-2. These growth factors are produced by the ureteric bud or metanephric
mesenchyme
(2,4,5,6). Since the initial discovery of EGF and its receptor, other growth factors that signal through the EGF receptor, i.e., the so-called EGF family of growth factors, have been identified. Heparin-binding EGF-like growth factor (HB-EGF), a relatively new member of this family, was originally purified from conditioned medium from U-937 cells, which were derived from a human histiocytic lymphoma (7). HB-EGF demonstrates potent mitogenic functions in several types of epithelial cell lines, including renal and intestinal epithelial cells, through activation of the intrinsic tyrosine kinase of the EGF receptor (8,9,10). Mesenchymal cell-derived cells, such as mesangial cells, vascular smooth muscle cells, fibroblasts, and keratinocytes, can also be stimulated to proliferate (11,12,13,14). The mitogenic activity of HB-EGF is potentiated through binding to cell surface heparan sulfate proteoglycans (15). Similar to other EGF family members, HB-EGF is initially synthesized as a membrane-anchored precursor (proHB-EGF), which exists as a 206-amino acid transmembrane protein (29 kD) and undergoes processing to an 86-amino acid secreted protein (mature HB-EGF, 22 kD) in a protein kinase C (PKC)-dependent process (7,16,17). ProHB-EGF can contact and activate cognate receptors on adjacent cells through binding to EGF receptors, establishing a mode of stimulation known as juxtacrine stimulation (10,18). During the process of organ development, juxtacrine stimulation can be an efficient method to selectively deliver mitogenic signals to adjacent cells. We recently reported that, when rat renal epithelial cells (NRK 52E, a cell line with distal nephron characteristics) were stably transfected with proHB-EGF, they proliferated and underwent tubulogenesis (19).
Using UBC, we demonstrate here that HB-EGF can induce branching tubulogenesis in three-dimensional collagen gel cultures. Differences in morphogenesis induced by HB-EGF with and without cooperation with TGF-ß or proHB-EGF were also examined.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(Genzyme,
Cambridge, MA), 100 U/ml ampicillin, and 100 µg/ml streptomycin.
ProHB-EGF-transfected UBC (UBCproHB-EGF) or empty
vector-transfected UBC (UBCvector) were continuously grown in the
aforementioned medium supplemented with geneticin (G418 sulfate, 400 µg/ml;
Life Technologies, Gaithersburg, MD).
Immortalized metanephric mesenchymal cells (MS-7 cells) were obtained from
an 11.5-d embryonic kidney from a homozygous mouse transgenic for H-2Kb-tsA
(21). The collected embryonic
kidney exhibited two ureteric bud branches. After trypsinization, mesenchymal
caps were carefully isolated with a thin needle. After the cells were
triturated, suspended, and placed on collagen-coated dishes, they were grown
in minimal essential medium with 10% FCS and interferon-. The
mesenchymal phenotype was confirmed by positive immunofluorescence for
vimentin and absent immunofluorescence for Dolichos biflorus lectin,
cytokeratin, cadherin, and ZO1, as described previously
(22). After six passages, the
cells were used for experiments.
Production of Collagen Gels
Five volumes of type I collagen solution (Cellmatrix IA; Nitta Zelatin
Inc., Osaka, Japan) were mixed with 4 vol of 2.5-fold concentrated DMEM and 1
vol of 0.05 M NaOH with 2.2% NaHCO3 and 200 mM Hepes, followed by
incubation at 37°C. Cells (105 cells/six-well plate) were
resuspended in ice-cold gel mixture. In particular experiments, the gel
mixture was supplemented with human recombinant soluble HB-EGF (rsHB-EGF) (R
& D Systems, Minneapolis, MN) and/or human recombinant TGF-ß1 (R
& D Systems). The solution was dispensed into plastic culture dishes
(Falcon six-well plates; Becton-Dickinson, Franklin Lakes, NJ) in 3-ml
aliquots and was allowed to solidify for 5 to 10 min at 37°C. The collagen
gels were overlaid with DMEM containing 1% FCS and were incubated at
37°C.
cDNA and Vectors
Mouse membrane-anchored HB-EGF cDNA was prepared according to the
previously described method
(10). In brief, mRNA was
extracted from kidneys from BALB/C mice (weighing 30 to 40 g) that had been
treated with gentamicin. A 688-bp fragment representing the mouse
membrane-anchored proHB-EGF coding region was amplified by PCR from the mRNA,
using the primer pair of 5'-GGACCATGAAGCTGCTGCCGTCG-3' and
5'-TAACGAACCACTGTCTCAGAAGT-3'
(23). The PCR amplification
was performed with 30 cycles of 94°C for 30 s, 72°C for 60 s, and
58°C for 45 s. The PCR product was then sequenced to confirm identity with
the previously published sequence data for the mouse membrane-anchored
proHB-EGF coding region (23).
The PCR fragment was initially ligated into pPCRII vector (Invitrogen, San
Diego, CA). A HindIII/NotI-digested cDNA fragment was then
ligated into an eukaryotic expression vector (pRc/CMV plasmid; Invitrogen).
Rat TGF-ß cDNA was a gift from Dr. Enyu Imai (The First Department of
Internal Medicine, Osaka University Faculty of Medicine, Osaka, Japan).
Transfection
Cultured UBC were stably transfected with proHB-EGF/pRc/CMV by the use of
lipofectin (Life Technologies) and were subsequently selected by continuous
growth in geneticin (G418, 400 µg/ml; Life Technologies). After five
passages, 50 individual clones were isolated and screened for proHB-EGF
expression by dot Northern blot analysis, using a 32P-labeled,
EcoRI-digested, membrane-anchored HB-EGF cDNA fragment. Finally, five
clones with high expression levels were identified. Two of these, UpHB 7 and
UpHB 14, were used in this study. As a control, UBC were transfected with
pRc/CMV plasmid alone and maintained in the presence of geneticin.
RNA Isolation and Northern Blot Analysis
Collagen gels containing UBC were homogenized in 2 vol of Isogen solution
(Nippon Gene, Tokyo, Japan), followed by RNA extraction as described elsewhere
(24). RNA from monolayer
cultured cells on plastic dishes was also collected and subjected to Northern
blot analysis, according to previously described methods
(10,25).
Aliquots of total RNA (each containing 15 to 20 µg) were subjected to
electrophoresis, transferred to S & S Nytran nylon membranes (Schleicher
& Schuell, Keene, NH), and fixed to the membranes by exposure to
ultraviolet light (CL-1000 ultraviolet crosslinker; Funakoshi Co., Tokyo,
Japan). The membranes were prehybridized at 42°C in a solution containing
30% formamide, 5x SSC, 5x Denhardt's solution (Wako Pharmaceutical
Co., Osaka, Japan), and 100 µg/ml sonicated salmon sperm DNA (Invitrogen).
The blots were hybridized overnight at 42°C with 0.5 to 1.0 cpm/ml
32P-labeled cDNA. After two washes each with 2x SSC for 15
min at room temperature, one wash with 1x SSC/0.1% sodium dodecyl
sulfate (SDS) for 15 min at 65°C, and finally one wash with 0.2x
SSC/0.1% SDS for 15 min at 65°C, the membranes were exposed to Kodak
X-Omat AR film at -70°C, with an intensifying screen. The blots were
stripped and reprobed with a human glyceraldehyde-3-phosphate dehydrogenase
cDNA (Clontech, Palo Alto, CA), using the conditions described above. mRNA
levels were quantitated by densitometry and normalized to
glyceraldehyde-3-phosphate dehydrogenase expression.
Antibodies
Goat anti-mouse HB-EGF antibody was purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). This antibody, which was raised against a peptide
corresponding to an amino acid sequence located at the carboxyl terminus of
proHB-EGF of mouse origin, can recognize both proHB-EGF and the soluble form
of HB-EGF and also crossreacts with rat HB-EGF (according to the information
provided by the manufacturer). The following antibodies were also used: mouse
monoclonal antibody to the EGF receptor (Transduction Laboratories, Lexington,
KY), mouse monoclonal antibody to phosphotyrosine (clone PY20; Transduction
Laboratories), mouse monoclonal antibody to phosphoinositide-3-kinase
(PI-3-kinase) (Transduction Laboratories), rabbit anti-TGF-ß antibody (R
& D Systems), rabbit antibody to matrix metalloproteinase-1 (MMP-1)
(Chemicon, Temecula, CA), goat antibody to MMP-2 (Santa Cruz Biotechnology),
rabbit antibody to rat aquaporin-2
(26), alkaline
phosphatase-conjugated goat anti-mouse IgG (EY Laboratories, San Mateo, CA),
rabbit antigoat IgG antibody (Cappel Research Products, Durham, NC) labeled
with FITC, and goat anti-rabbit IgG antibody (Cappel).
Immunoblotting
UBC and MS-7 cells cultured in the medium described above were solubilized
in RIPA buffer (1% Nonidet P-40, 150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 50 mM
NaF, 100 µM Na3 VO4, 5 mM
ethylenedia-minetetraacetate, 0.1% SDS, 0.5% sodium deoxycholate, 1%
aprotinin). The protein concentration in the lysates was measured by using a
bicinchoninic acid protein assay kit (Pierce, Rockford, IL). The lysates,
containing equal amounts of protein (300 µg), were incubated with the
primary antibody, followed by incubation with Pansorbin (Calbiochem, La Jolla,
CA). After electrophoresis on 6% SDS-polyacrylamide gels, the proteins were
transblotted onto Immobilon-P membranes (Millipore Japan, Yonezaya, Japan) in
transfer buffer (25 mM Tris-HCl, pH 8.5, 192 mM glycine, 20% methanol) at 30 V
for 16 h. After incubation at room temperature in Tris-buffered saline (10 mM
Tris-HCl, pH 7.4, 150 mM NaCl) containing 3% bovine serum albumin, the
membranes were incubated again with the primary antibody (the antibody used
for immunoprecipitation) for 2 h at room temperature, followed by incubation
with the appropriate secondary antibody conjugated with alkaline phosphatase.
The reaction products were detected using a 5-bromo-4-chloro-3-indolyl
phosphate/nitro blue tetrazolium phosphatase substrate system (Kirkegaard
& Perry Laboratories, Gaithersburg, MD). Phosphorylation of the EGF
receptor in UBC was demonstrated by immunoprecipitation with anti-EGF receptor
antibody at the first step described above and detection with
anti-phosphotyrosine antibody on the blotted membranes at the second step.
Rat embryonic kidneys (embryonic days 14 and 18) and kidney tissues after birth (1 d, 10 d, and 12 wk after birth) were collected and homogenized with a Dounce homogenizer in RIPA buffer. After centrifugation, the supernatants were dialyzed against phosphate-buffered saline (PBS) and freeze-dried. The materials were then dissolved in RIPA buffer. The fractions (300 µg of protein) were subjected to immunoprecipitation with anti-HB-EGF antibody and then to Western blotting, as described above.
Determination of [3H]Thymidine Incorporation
Cells were cultured in 24-well plates until confluent and were incubated in
FCS-free DMEM for 72 h before the addition of rsHB-EGF (10-7 M).
Subsequently, 1 µCi/ml [3H]thymidine was added and the cells
were incubated for an additional 4 h. After two washes with ice-cold PBS, the
cells were incubated for 30 min on ice with 10% TCA and were rinsed with
ethanol. The acid-precipitated pellets were dissolved in 0.25 M NaOH/0.1% SDS
and quantitated by scintillation counting.
Immunocytochemical Staining
Rat kidney tissues were fixed in paraformaldehyde-lysine periodate, treated
sequentially with 10, 15, and 20% sucrose in PBS, and finally embedded in
paraffin solution. UBC were cultured in six-well plastic dishes, coated with
CELL-TAK cell and tissue adhesive solution (Collaborative Biomedical Products,
Bedford, MA) until confluent, and then fixed in 1% paraformaldehyde/PBS for 15
min. The rat kidney tissue sections or cultured cells were examined by
indirect immunofluorescence or immunoperoxidase staining methods, using the
antibodies described above, as previously reported
(27,28).
As controls, tissue sections or cells were incubated with nonimmune goat or
rabbit serum, followed by incubation with the appropriate secondary antibody,
or were incubated with the secondary antibody alone. These controls yielded
completely negative results.
Electron Microscopic Observations
Collagen gels containing UBC were fixed in 2.5% glutaraldehyde/0.1 M sodium
cacodylate buffer (pH 7.4) for 12 h. After extensive washing with cacodylate
buffer, collagen gels were scraped from the culture dishes, cut into 3-mm
x 3-mm sections, postfixed with 1% osmic acid/PBS (pH 7.2) for 60 min,
and stained with uranyl acetate in 50% ethanol. The stained sections were then
dehydrated in graded ethanol and embedded in Epon 812 (Taab, Aldermaston, UK).
Ultrathin sections (600 to 900 
) were prepared
and examined by electron microscopy.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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EGF Receptor Phosphorylation, PI-3-Kinase Expression, and Cell Growth
Induced by rsHB-EGF
UBC were cultured in the presence of rsHB-EGF (10-7 M). In
Western blot analyses, the UBC lysate immunoprecipitates exhibited EGF
receptor phosphorylation (Figure
2A) and PI-3-kinase upregulation
(Figure 2B). After exposure to
rsHB-EGF, UBC exhibited an approximately threefold increase in
[3H]thymidine incorporation
(Figure 2C).
|
Branching Tubulogenesis of UBC Treated with TPA and Cultured in Type
I Collagen Gels
After stimulation with TPA for 30 min, UBC were immersed in type I collagen
gels in medium containing 1% FCS. The cells developed short branches by day 1
and extended into the surrounding collagen matrix, exhibiting tubular
structures with varied diffuse branches, by day 2 or 3
(Figure 3, a to d). When
TPA-treated UBC were cultured in the presence of neutralizing anti-HB-EGF
antibody, branching differentiation was partially inhibited
(Figure 3e). Northern blot
analysis of mRNA from UBC grown in collagen gels demonstrated that HB-EGF mRNA
expression was upregulated after treatment with TPA and peaked in the
subsequent 24 to 48 h (data not shown), concomitant with the morphologic
changes of branching cord elongation.
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Branching Tubulogenesis of UBC Grown in Collagen Gels Supplemented
with rsHB-EGF
When UBC were cultured in collagen gels supplemented with rsHB-EGF (30
ng/ml) in medium containing 1% FCS, they began to develop short branching
cords by day 1 and gradually formed abundant arborization by days 2 to 4
(Figure 4A, a and b). The
structures resembled those found in UBC stimulated with TPA. Expression of
MMP-1 was upregulated in these cells
(Figure 4B). Addition of
neutralizing anti-HB-EGF antibody to collagen gels inhibited tubulogenesis and
branching (Figure 4A, c).
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Tubulogenesis of UBC Transfected with Membrane-Anchored HB-EGF
(ProHB-EGF)
To investigate the role of proHB-EGF (the membrane-anchored form of HB-EGF)
in the process of branching tubulogenesis, UBC constitutively expressing
proHB-EGF (UBCproHB-EGF) were established and cultured in collagen
gels. HB-EGF expression at the mRNA and protein levels by
UBCproHB-EGF was confirmed by Northern blotting and immunodetection
(Figure 5A). Because cleavage
of proHB-EGF to its soluble form occurs within 60 min after activation of the
PKC pathway and membrane-anchored proHB-EGF is recovered within the subsequent
6 to 12 h (29), we incubated
UBCproHB-EGF with 0.1 mM TPA for 30 min. The cells were then
extensively rinsed with medium containing 2 M NaCl, to remove soluble HB-EGF
trapped by cell surface heparan sulfate proteoglycans. UBCproHB-EGF
cultured in monolayers were stained with anti-HB-EGF antibody. After plating
for 12 h, proHB-EGF protein appeared on the surface membrane, especially at
cell-cell contact sites (Figure
5B).
|
After incubation with TPA and washing with medium containing 2 M NaCl, UBCproHB-EGF were immersed in collagen gels and cultured in medium containing 1% FCS. The controls, i.e., wild-type UBC and UBCvector, were treated in a similar manner. UBCproHB-EGF formed short epithelial branching cords during the first 12 h and then elongated, exhibiting linear tubular structures within 24 to 36 h (Figure 6). After 48 to 96 h, they formed branches. Compared with UBC cultured in the presence of TPA or rsHB-EGF, UBCproHB-EGF exhibited longer tubules with minimal branches, accompanied by well organized luminal structures. Electron microscopic analyses revealed luminal and basolateral polarization, similar to the findings presented in Figure 7D. The cells exhibited microvillus-rich apical surfaces. Aquaporin-2, which is normally present in collecting ducts (26), was immunocytochemically detected in the UBC (Figure 5C). Wild-type UBC and UBCvector in culture exhibited only clusters of round cells. There were no differences in the levels of TGF-ß mRNA expression among the three cell types (data not shown).
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UBC Culture with rsHB-EGF and TGF-ß
When UBC were cultured in collagen gels containing rsHB-EGF (30 ng/ml) and
recombinant TGF-ß1 (2 ng/ml), they developed single branching cords by 12
h, which subsequently differentiated to long tubules with little branching
(Figure 7, A to D). Finally,
the UBC formed straight tubule-like structures with less branching and well
organized lumina, resembling structures that developed in
UBCproHB-EGF. UBC cultured with rsHB-EGF alone, without recombinant
TGF-ß1, formed abundant short branches
(Figure 7E). Addition of
TGF-ß1 alone to UBC did not induce tubulogenesis. To determine the source
of TGF-ß, Northern blot analysis of mRNA from UBC and MS-7 cells was
performed. TGF-ß mRNA and protein expression was detected in mesenchymal
cells (MS-7 cells) but not in UBC (Figure
8). TGF-ß1 suppressed the expression of MMP-1 in
rsHB-EGF-stimulated UBC (Figure
4B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IGF-1, GDNF, and bone
morphogenic protein-7, have been demonstrated to induce epithelial cell
tubulogenesis. On the basis of accumulating evidence
(1,2,3),
it is likely that the balance between growth-promoting factors (e.g.,
HGF), branching-promoting factors (e.g., GDNF), and growth-inhibiting
factors (e.g., TGF-ß) regulates branching tubulogenesis.
Available data also suggest redundancy in these regulatory pathways, because
genetically engineered mice that lacked HGF, c-Met (HGF receptor),
TGF-, or IGF-1 exhibited mostly normal metanephric development
(31,32,33).
EGF receptor-knockout mice developed dilated collecting ducts and impaired
renal function (34).
Tyrphostin AG1478, an inhibitor of EGF receptor tyrosine kinase, blocked
tubulogenesis of mIMCD3 cells growin in collagen gels
(5). Culture studies using
embryonic kidney rudiments demonstrated that sulfated glycosaminoglycans were
upregulated during nephron development
(35). Furthermore, removal of
these molecules, although it did not affect c-met expression,
inhibited ureteric bud growth and branching
(35), suggesting that ureteric
bud growth and morphogenesis are regulated by distinct signaling pathways
mediated via sulfated glycosaminoglycans. These data collectively indicated
that growth factors that belong to the EGF family, other than EGF or
TGF-, and have an affinity for sulfated molecules might be important
for collecting duct morphogenesis. Therefore, we examined the role of HB-EGF in ureteric bud growth and branching. In the initial study, we noted that HB-EGF protein was immunocytochemically evident in the ureteric bud and that HB-EGF mRNA expression was upregulated in cultured UBC after TPA stimulation. The exogenous addition of HB-EGF to quiescent UBC stimulated cell proliferation, with tyrosine phosphorylation of the EGF receptor. Next, we observed that, after TPA incubation, UBC formed tubule-like structures in collagen culture. Similar morphologic features were observed when UBC were grown in the presence of the soluble form of HB-EGF. As previously demonstrated by Cantley et al. (36), these cells exhibited activation of the intracellular signaling molecule phosphoinositide, as well as upregulation of matrix-degrading enzymes. Branching of UBC stimulated with TPA or soluble HB-EGF resembled that of MDCK cells grown in the presence of HGF (6, 36, 37). Previous studies (6, 36, 38,39,40), together with the studies presented here, indicate that phosphorylation of tyrosine residues occurs through activation of either c-Met by HGF or EGF receptor by HB-EGF, which leads to stimulation of downstream signals such as PI-3-kinase. It seems that HGF and mature HB-EGF may share a common intracellular signaling pathway that leads to changes in cell shape and cell movement. Morphogenesis observed in the culture system of HGF-treated MDCK cells or mature HB-EGF-treated UBC was somewhat dissimilar from the early branching ureteric bud observed in vivo in embryonic kidneys, because those cultured cells formed short tubules with varied abundant branches. Therefore, we speculated that factors other than HGF or mature HB-EGF were needed to induce long nonbranching tubules.
We addressed the role of proHB-EGF (the membrane-anchored form of HB-EGF)
in collecting duct morphogenesis. It is known that cleavage of
membrane-anchored proHB-EGF to its soluble peptide is regulated by
PKC-dependent proteolysis. Maximal loss of cell surface proHB-EGF occurs
within 30 to 60 min after PKC activation, and membrane-anchored proHB-EGF is
restored within the subsequent 6 to 12 h
(29). We treated
UBCproHB-EGF with TPA, removed soluble HB-EGF, and then placed the
cells in collagen gels. ProHB-EGF reappeared at the cell surface membrane,
particularly at cell-cell contact sites. Without addition of any other growth
factors, the cells formed linear tubules with minimal branches. The
superficial structures of the cultured cells exhibited a strong resemblance to
the early branching ureteric bud of embryonic kidneys. In contrast, the
soluble form of HB-EGF induced varied diffuse growth, with branching. The
difference between the branching caused by soluble HB-EGF and that produced by
the membrane-bound form may be explained by juxtacrine activation, which is a
unique mode of action exhibited by proHB-EGF. In general, juxtacrine
activation has an advantage with respect to constant signaling exclusively to
adjacent cells. In our previous studies, proHB-EGF-transfected NRK 52E cells
(derived from rat distal nephron) exhibited TPA-dependent differentiation
in vitro, resulting in the formation of single tubule-like structures
(19). The cells exhibited
increased migration on the plates and remarkable changes in shape associated
with the rearrangement of F-actin and -tubulin.
UB-CproHB-EGF in this study or proHB-EGF-transfected NRK 52E cells
in our earlier study (10)
exhibited no differences in the levels of TGF-ß mRNA expression, compared
with wild-type or vector-transfected cells. Tubulogenesis mediated by
proHB-EGF is different from that mediated by HGF
(6,
36,
37) or soluble HB-EGF and is
also independent of TGF-ß mRNA expression. Therefore, it is speculated
that juxtacrine activation, which is a unique action of proHB-EGF, is related
to the formation of long tubules. In addition, our preliminary studies
suggested that proHB-EGF, compared with soluble HB-EGF, tends to promote cell
survival by inhibiting apoptosis
(41), which might facilitate
differentiation to form tubular structures in UBC. However, the precise
mechanisms involved in juxtacrine activation by membrane-anchored growth
factor, eliciting cellular morphogenesis responses, as distinct from
activation by the soluble form of the growth factor, are still unclear.
We observed that UBC cultured in the presence of both rsHB-EGF and TGF-ß exhibited long tubules with few branches, similar to those formed in UBCproHB-EGF. TGF-ß is a multifunctional peptide that exerts its effects in the suppression of cell proliferation, the stimulation of extracellular matrix production, and the suppression of MMP (42). In organ culture, addition of exogenous TGF-ß inhibits embryonic kidney differentiation (43). TGF-ß alone has no obvious effect on morphogenesis. However, in the presence of growth- or branch-facilitating factors, TGF-ß inhibits either tubulogenesis or branching or both, depending on the cell type (24). In this study, we observed that metanephric mesenchymal cells, but not UBC, produced TGF-ß in culture. TGF-ß suppressed the expression of MMP-1 in HB-EGF-stimulated UBC. Previous studies from other laboratories revealed that, in in situ hybridization analyses, TGF-ß mRNA signals were most prominent in the mesenchymal/stromal cells adjacent to branching tubules and TGF-ß protein expression seemed to be greatest near the ureteric bud (43, 44). In mIMCD3 cells grown in collagen gels in the presence of HGF, TGF-ß inhibited the expression of MMP-1 and urokinase and stimulated the synthesis of their inhibitors (24). Therefore, mesenchymal cells can be a source of TGF-ß. It seems that TGF-ß contributes to the formation of long linear tubules by inhibiting branching via modulation of the balance between matrix-degrading enzymes and their inhibitors.
In summary, these results indicate that HB-EGF may be one of the regulators involved in the localization of branches and determination of the stalk length between branches. The soluble form of HB-EGF can facilitate branching. In contrast, membrane-anchored HB-EGF can induce linear tubular structures with few branches, which resemble those that form in the presence of rsHB-EGF and TGF-ß. These data provide a basis for future research to produce highly differentiated tubules. The refinement of in vitro tubule culture techniques would make it possible to obtain artificial tubules for the treatment of developmental disorders of the kidney or acute tubular injury.
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Acknowledgments |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene have abnormal skin architecture, wavy
hair, and curly whiskers and often develop corneal inflammation.
Cell 73:249
-261, 1993[Medline]
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