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Regular Articles |
From the Laboratory of Cell Regulation andCarcinogenesis*
and Radiation BiologyBranch,
National Cancer Institute and theGenetics of Development and Disease Branch,
National Institute of Diabetes and Digestive and Kidney Diseases,National Institutes of Health, Bethesda, Maryland
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-irradiation. Six weeks later,
skin from KO mice showed significantly less epidermal acanthosis and
dermal influx of mast cells, macrophages, and
neutrophils than skin from WT littermates. Skin from irradiated KO mice
exhibited less immunoreactive TGF-ß and fewer myofibroblasts,
suggesting that these mice will have a significantly reduced fibrotic
response. Although irradiation induced no change in the
immunohistochemical expression of the TGF-ß type I receptor,
the epidermal expression of the type II receptor was lost after
irradiation whereas its dermal expression remained high. Primary
keratinocytes and dermal fibroblasts prepared from WT and KO mice
showed similar survival when irradiated, as did mice exposed to
whole-body irradiation. These results suggest that inhibition of Smad3
might decrease tissue damage and reduce fibrosis after exposure to
ionizing irradiation.
At the cellular level, TGF-ß affects virtually all stages of the chronic inflammatory and fibrotic disease process. It is a potent chemotactic factor for monocytes,6 neutrophils,7,8 mast cells,9,10 and fibroblasts,11 active at femtomolar concentrations. After recruitment of inflammatory cells into an area of insult, TGF-ß also activates neutrophils and induces macrophages to secrete cytokines, one of which is TGF-ß itself. This autoinduction of TGF-ß is important for recruitment of additional inflammatory cells and maintaining local elevated levels of TGF-ß.5 The TGF-ß produced by macrophages can then induce matrix production by fibroblasts. The sustained expression of TGF-ß is critical to the maintenance of the inflammatory and fibrotic response.
TGF-ßs signal through transmembrane receptors with intrinsic serine/threonine kinase activity. Binding of ligand to these heteromeric receptors induces carboxyl-terminal serine phosphorylation of a set of cytoplasmic signal-transducing proteins collectively referred to as "Smad" proteins. After activation/phosphorylation, pathway-specific Smad proteins (Smad2 and Smad3 for TGF-ß signaling) heterodimerize with the common mediator Smad4 and this complex translocates to the nucleus to regulate expression of specific target genes.12 Smad7 (an inhibitory Smad) can disrupt signal transduction by preventing phosphorylation of Smad2 or Smad3.13,14 Mitogen-activated protein kinase pathways, induced by TGF-ß or by other inputs, as well as protein kinase C activation also modulate TGF-ß signaling by altering phosphorylation of Smads at sites other than the C-terminal serines phosphorylated by ligand-activated receptors.15,16
Smad2 and Smad3, although highly homologous, have distinct modes of action. Smad3 regulates target gene activity directly by binding to DNA,17 whereas Smad2 activates transcription instead by binding to other DNA-binding transcription factors to modulate their activity.18 The different DNA-binding characteristics of Smad2 and Smad3 result in regulation of distinct sets of target genes. The distinct activities of Smad2 and Smad3 are evidenced by the finding that targeted deletion of the Smad2 gene results in early embryonic lethality19-21 whereas mice null for Smad3 are viable for up to 8 months.22-24 Studies using fibroblasts derived from embryos null for either Smad2 or Smad3 show that TGF-ß1-mediated autoinduction and induction of c-fos are Smad3-dependent.25 Many genes, such as the ECM proteins collagen type I and type VII, contain AP-1 binding sites in their regulatory regions, and their induction by TGF-ß has been shown to be Smad3-dependent.26-29
Characterization of the basal phenotype of Smad3-null mice, as well as studies of incisional wound healing in these mice suggest that Smad3 has an important function in both inflammation and fibrosis in vivo. Smad3-null mice die from defects in mucosal immunity suggestive of defects in neutrophil chemotaxis and consistent with the observation that the chemotactic response of Smad3-null neutrophils to TGF-ß is impaired both in vivo and in vitro.24 Cutaneous incisional wounds in Smad3-null mice show reduced influx of inflammatory cells, decreased accumulation of matrix, and enhanced re-epithelialization.30 This results, in part, from the impaired ability of Smad3-null macrophages to respond to TGF-ß with chemotaxis and autoinduction; reduced levels of TGF-ß in the granulation tissue then result in less ECM production. Additionally, Smad3-null keratinocytes lose their ability to be growth inhibited by TGF-ß which contributes to the enhanced epithelialization of the wounds. Chronic inflammatory and fibrotic diseases share many common features with wound healing, except that the fibrotic process does not resolve. Because loss of Smad3 interferes with the effects of TGF-ß on chemotaxis and autoinduction in inflammatory cells, and because induction of many ECM genes by TGF-ß are also dependent on Smad3, we hypothesized that mice null for Smad3 will be resistant to chronic inflammation and fibrosis in which TGF-ß has been shown to be involved.5
Radiation fibrosis has features common to other fibrotic diseases, including the involvement of TGF-ß in its pathogenesis.31 Ionizing radiation exposure frequently induces damage in the skin and underlying subcutaneous tissue. Early lesions are characterized by erythema, dry and moist desquamation, and ulceration resulting from the infiltration of inflammatory cells and increased vascularization. As a result of ionizing irradiation, the epidermis may be hyperplastic or atrophic. Likewise, activated dermal fibroblasts are characterized by the appearance of cytoskeletal proteins similar to those involved in wound contraction, such as smooth muscle actin, and a deposition of a fibrous matrix that is abnormal in both quantity and quality. These fibrotic sequelae are an unwanted complication of radiotherapy and severely impact on a patient’s quality of life. Biopsies removed surgically from fibrotic lesions 6 months to 20 years after radiotherapy show enhanced expression of mRNA for collagen type I and type III and TGF-ß1.31 Similarly, in an experimental model of cutaneous radiation in the pig, TGF-ß1 mRNA was increased at 1 to 12 months after irradiation32 and immunoreactive TGF-ß1 was localized to myofibroblasts, endothelial cells, and the collagen matrix. Irradiation of mouse skin also shows increased expression of TGF-ß1 mRNA from 6 hours to 9 months after irradiation.33 Irradiation of other tissues such as lung,34 intestine,35 bladder,36 and liver37 is also associated with increased TGF-ß expression.
Because elevated levels of TGF-ß are associated with
radiation-induced inflammation and fibrosis, and because Smad3-null
mice show reduced inflammation, TGF-ß1, and matrix accumulation after
wounding, we hypothesized that loss of Smad3 will decrease inflammation
and subsequent fibrosis induced by irradiation. To test this
hypothesis, we exposed the skin of Smad3+/+ (WT), +/- (Het), and -/-
(KO) mice to high doses of 
-irradiation. Here we report that 6 weeks
after irradiation skin from KO mice shows significantly less epidermal
acanthosis, dermal inflammation, and immunoreactive TGF-ß than does
skin from WT mice.
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Materials and Methods |
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Smad3ex8/ex8 mice were generated by targeted disruption of the Smad3 gene by homologous recombination. Targeted embryonic stem-cell clones were microinjected into C57BL/6 blastocysts to obtain germline transmission. Mice heterozygous for the targeted disruption were intercrossed to produce homozygous offspring.24
Whole-Body Irradiation
The protocol used in this study was institutionally approved and in accordance with guidelines of the Institute of Laboratory Animal Resources, National Research Council. Animals were exposed to whole-body irradiation using a 137Cesium Gamma Cell 40 (Nordion Int. Inc., Kanata, Ontario) irradiator that had been calibrated with thermoluminescent dosimetry chips (Bicron, Inc., Solon, OH) planted in phantom Plexiglas mice. Animals were irradiated between 7 to 9 weeks after birth. Smad3 WT, Het, and KO mice were placed in circular Lucite containers with holes for ventilation (up to five animals). The container was positioned within the irradiator and exposed for varying times to deliver the desired dose (single radiation doses ranging from 6 to 10 Gy at a dose rate of 0.98 Gy/minute). After irradiation, mice were removed from the container and returned to the cages in which they were housed (five animals/cage) in a climate-controlled environment with free access to food and water. If at any time an animal was unable to acquire food or water because of illness, the animal was euthanized per protocol. Mice were observed daily for survival.
Local Irradiation
Experiments also involved local irradiation to the leg or flank. For these studies animals (without anesthetics) were placed in customized Lucite jigs that allow for immobilization and selective irradiation of the leg or skin of the flank. Special clamps held the skin without decreasing blood flow to the treatment area as determined previously by laser Doppler studies.38 Single radiation doses ranging from 30 to 60 Gy were delivered by a Therapax DXT300 X-ray irradiator (Pantak, Inc., East Haven, CT) using 2.0-mm Al filtration (300 kVp) at a dose rate of 1.9 Gy/minute. Special care was taken to avoid irradiation of other body parts by using lead shields specifically designed as a part of the jigs. In addition to WT, Het, and KO mice, four mice each of strains C57BL/6nCr, Tac:N:NIHS-BCFBR, 129S6/SvEv-ATM<tm1awb>, and WBB6F1/J KIT/KITW-v (mast cell deficient)39 also received hind leg irradiation. After irradiation, the animals were placed in cages as indicated above and observed daily for 5 to 6 weeks at which time animals were euthanized and skin was removed from the hind legs or flank and transferred to 10% neutral buffered formalin for 24 hours. After three washes in 70% ethanol, tissues were processed for paraffin embedding.
Quantitative Histopathology and Morphometry
Formalin-fixed, paraffin-embedded tissues were cut at 5 µm and sections were stained with hematoxylin and eosin (H&E), Geimsa (to identify neutrophils), or low pH toluidine blue (to identify mast cells). The numbers of neutrophils and mast cells were counted in 5 400x magnification fields/skin section.
Macrophages, blood vessels, and myofibroblasts were identified by staining with rat anti-mouse Mac-3 (BD PharMingen, San Diego, CA), rat anti-mouse CD31(PECAM-1) (BD PharMingen), and mouse anti-smooth muscle actin Ab-1 (NeoMarkers Inc., Fremont, CA), respectively. Staining was performed using the Optimax Plus 2.0 Automated Cell Staining System with research software (BioGenex, San Ramon, CA). For anti-Mac-3 staining, tissue sections were deparaffinized, treated with 1% H2O2 and nonspecific protein binding was blocked for 1 hour with a solution containing 1% bovine serum albumin and 5% rabbit serum. Sections were incubated for 2 hours at 37°C with anti-Mac-3 (2.5 µg/ml) in Tris-buffered saline, pH 7.4/1% bovine serum albumin. Antigen-antibody complexes were detected using the Vectastain Elite rat ABC peroxidase kit (Vector Laboratories, Burlingame, CA) according to the manufacturer’s instructions. After 30 minutes of incubation with biotinylated secondary antibody followed by a 30-minute incubation with ABC reagent, a 5-minute reaction with diaminobenzidine/H2O2 was used to detect the bound peroxidase. Staining with anti-CD31 was similar except that sections were also pretreated for 30 minutes with 0.25% trypsin (Sigma Chemical Co., St. Louis, MO) and the primary antibody incubation was performed overnight at 4°C at 6.5 µg/ml. Staining for anti-smooth muscle actin was performed with the Vector M.O.M. kit (Vector Laboratories) according to the manufacturer’s instructions using a 2-hour incubation with 0.5 µg/ml of smooth muscle actin IgG. For all staining experiments isotype-matched normal IgG at the same concentration as the primary antibody was used as a negative control. The numbers of macrophages, blood vessels, and myofibroblasts were counted in 5 400x magnification fields/skin section.
Quantitation of epidermal thickness was done by capturing images of H&E-stained sections (x400 magnification) from a Zeiss Axioplan microscope using an MTI 3 charge-coupled device camera. On each section three areas of interfollicular acanthosis were identified by eye and the thickness of the epidermis from the outer edge to the epidermal-dermal interface was measured using ImagePro 2.0 software.
Quantitative results are expressed as mean ± SE mean. Significant intergroup differences were determined by applying the two-sample assuming unequal variance t-test.
TGF-ß Ligand and Receptor Levels
Immunoreactive TGF-ß1, TGF-ß2, and TGF-ß3, as well as the type I (RI) and type II (RII) receptors were localized to tissue sections using a protocol similar to that described above for staining with the Mac-3 antibody with the following modifications. Sections were pretreated for 30 minutes with 1 mg/ml of bovine testicular hyaluronidase (Sigma) for TGF-ß ligand staining, the primary antibody incubation was performed for 2 hours at room temperature, and a Vectastain Elite rabbit ABC peroxidase kit was used. The primary antibodies used to detect TGF-ß1 were IgG fractions of LC 1-30-1 (6 µg/ml) and CC 1-30-1 (0.2 µg/ml), which recognize intracellular and extracellular TGF-ß1, respectively.40 Affinity-purified anti-TGF-ß2 (catalog no. sc-90; Santa Cruz Biotechnology, Santa Cruz, CA) and anti-TGF-ß341 were used at 0.8 µg/ml and 4 µg/ml, respectively. RI and RII were detected with antibodies from Santa Cruz Biotechnology (catalog no. sc-398 and sc-220) that were used at 4 and 0.8 µg/ml, respectively.
Preparation of Keratinocytes and Fibroblasts
Epidermal keratinocytes were isolated from polymerase chain reaction-genotyped Smad3 WT and KO newborn mice by standard methods42 and were cultured in Eagle’s minimal essential medium/8% Chelex-treated fetal bovine serum/0.05 mmol/L CaCl2 with antibiotics. Fibroblasts were isolated by collagenase (Life Technologies, Inc., Grand Island, NY) digestion of the pooled dermal layers from the newborn WT and KO pups according to Lichti and colleagues43 and were cultured in Dulbecco’s modified Eagle’s medium/10% fetal bovine serum/1% Pen-Strep.
Cell Survival Assay
Epidermal keratinocytes were plated and cultured for 2 days before
being exposed to 0, 5, or 10Gy of -irradiation from a
60Co source. Cells were cultured for an
additional 48 hours at which time cells were trypsinized and counted
using a hemocytometer after trypan blue staining. The number of
surviving unirradiated cells was set as 100% and the surviving
irradiated cells were referenced to the unirradiated cells.
Dermal fibroblasts (passage 2) isolated from WT and KO mice were
removed from the tissue culture flask with 0.05%
trypsin-ethylenediaminetetraacetic acid (Life Technologies, Inc.) and
divided into six equal aliquots. One aliquot was exposed to each of
0.5, 2, 5, and 10 Gy of -irradiation, whereas two aliquots were left
unirradiated. Equal numbers of cells from each aliquot were plated into
35-mm dishes and allowed to grow for 5 days and then were rinsed,
trypsinized, and the number of surviving cells in each dish determined
with a Coulter counter. The number of surviving unirradiated cells was
set as 100% and the percentage of surviving irradiated cells was
compared to this.
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Based on our previous studies demonstrating that Smad3-null mice
show accelerated healing of incisional wounds,30
we
hypothesized that loss of Smad3 might improve healing or protect
against tissue damage resulting from other types of insults. To test
this hypothesis, the hind legs of a group of WT, Het, and KO mice were
exposed to 50 Gy of -irradiation. Animals were sacrificed 5 weeks
after irradiation to preclude losing KO animals to opportunistic
infections resulting from defects in T-cell activation and mucosal
immunity.24
Although this time point was too early to
observe fibrosis of the dermis or possibly also the underlying muscle
tissue, a striking difference in the histological appearance of the
skin between WT and KO animals was noted. Compared to unirradiated skin
(Figure 1A)
, the irradiated WT skin
(Figure 1C)
shows erosion of the epidermis with formation of an ulcer
base, along with a dense, compacted dermis with a marked increase in
cellularity. In contrast, the irradiated KO skin (Figure 1B)
, exhibits
only moderate acanthosis, hyperkeratosis, and keratin whorls with less
dermal compaction and inflammation. Table 1
shows the qualitative analysis of the
histological appearance of the skin of the legs 5 weeks after
irradiation. At this time point the epidermis has begun to heal from
the most severe damage observed (moist desquamation at peaking at 
25
days). At 35 days the epidermal damage in the KO mice (Figure 1B)
is on
average less severe (normal epidermis and moderate acanthosis) than
that in WT animals (Figure 1C)
in which a higher percentage of animals
show more severe damage (ulceration and severe acanthosis) (Table 1)
.
Samples from Het mice have an intermediate histological appearance. The
epidermal thickness of the restricted set of samples that were not
ulcerated showed no significant differences among the genotypes because
of the severity of the damage (data not shown).
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. The severity of
inflammation was graded based on the numbers of inflammatory cells in
the dermis and on the density of the dermal matrix. In quantitative
studies there were no significant differences in numbers of dermal
neutrophils or macrophages between WT and KO mice, however there were
significant differences in the numbers of mast cells (112 ± 11
for WT versus 60 ± 3 for KO, P <
0.0.001 per five high-power fields). Because the Smad3-null mice are carried on a mixed background of three strains, we irradiated legs of mice of each individual strain (C57BL/6nCr, Tac:N:NIHS-BCFBR, and 129S6/SvEv-ATM<tm1awb>) to test whether any of these strains might show increased resistance to irradiation. Irradiated skin from all three strains showed an acanthotic epidermis and severe dermal inflammation (data not shown). This assured that the protective effect seen in the KO animals is not the result of increased representation of a radio-resistant strain.
To obtain a greater area of irradiated skin for analysis, in subsequent
experiments the site of irradiation was changed to the flank skin,
again with care to shield the rest of the mouse from systemic
irradiation. Additionally, the dose of -irradiation was decreased to
30 Gy to reduce ulceration of the WT skin and to facilitate
quantitative analysis of the protective effects associated with loss of
the Smad3 gene. Thirty Gy caused less damage to the skin resulting in
only acanthosis of the WT epidermis at 6 weeks with none of the
ulceration that had been observed at the 50-Gy dose. The 30-Gy dose
also caused moist desquamation that peaked at 30 days, but had
resolved before the time of analysis. Again, the epidermis of skin from
irradiated WT mice (Figure 1, E and G)
is more hyperplastic than that
from KO mice and is characterized by hyperkeratosis, parakeratosis, and
keratin whorls that are absent from irradiated KO skin (Figure 1, F and H)
. Quantitation of the interfollicular epidermal thickness is shown in
Figure 2
. Skin from nonirradiated WT and
KO mice exhibits similar epidermal thickness before irradiation,
whereas the fold-increase after exposure to 30 Gy of irradiation is
8.0-, 4.8-, and 2.4-fold for WT, Het, and KO skin, respectively.
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shows that there are
approximately twofold more mast cells, macrophages, and neutrophils
present in the dermis of irradiated skin from WT mice as compared to KO
mice. Additionally, there are 70% more blood vessels and
myofibroblasts in the irradiated skin from WT animals than from KO
animals. Interestingly, the numbers of mast cells in the dermis of KO
mice remain unchanged after irradiation (Table 3)
, whereas irradiation induces an
increase in numbers of mast cells in the dermis of WT mice. There is a
greater fold-induction of neutrophils after irradiation in KO mice as
compared to WT animals, whereas the fold-induction of macrophages is
similar in both genotypes (Table 3)
. These data suggest that in dermal
inflammation induced by irradiation, the chemotaxis of mast cells and
neutrophils into the irradiated area may be more dependent on a
Smad3-mediated mechanism than is macrophage chemotaxis. However, the
basal levels of macrophages in unirradiated KO skin are considerably
reduced compared to those in WT skin. Additionally, the greater
increase in myofibroblasts in WT mice after irradiation compared to KO
mice, suggests that Smad3 may also play a role in the recruitment of
fibroblasts in response to TGF-ß.11
Conversion of
quiescent fibroblasts to activated myofibroblasts is also thought to be
a TGF-ß-dependent process,44-46
but the role of Smad3
is not known.
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, suggesting that the absence of mast cells did not protect
against radiation-induced injury despite the fact that mediators
released from mast cells have been proposed to promote epidermal
hyperplasia.47 TGF-ß Ligand and Receptor Expression in Irradiated Skin
Because TGF-ß1 levels have been observed to increase in tissues
after injury or in areas showing inflammation,4,5
and
because cells that are null for Smad3 are unable to autoinduce
TGF-ß1,25
we compared the intensity of the
immunohistochemical staining of the three TGF-ß isoforms in
irradiated flank skin from WT and KO mice. A summary of the changes in
immunoreactivity of the different TGF-ß isoforms in WT and KO skin
after irradiation is shown in Table 4
.
Two different antibodies were used for localization of TGF-ß1, LC
1-30-1 and CC 1-30-1, which recognize intracellular TGF-ß1 and
TGF-ß1 associated with ECM, respectively.40
There is
significantly more immunoreactive TGF-ß1 associated with the ECM in
the dermis of irradiated WT skin as compared to irradiated skin from KO
mice (Figure 3, A and B)
. Figure 3B
demonstrates that even in an area exhibiting epidermal hyperplasia in a
KO mouse, there is very little immunoreactive TGF-ß1 in the
underlying dermis (Figure 3B
, arrow). Additionally, keratinocytes in
the irradiated epidermis and inflammatory cells in the dermis show more
staining for intracellular TGF-ß1 in WT as compared to KO mice
(Figure 3, C and D)
. Regardless of which TGF-ß1 antibody is used,
there is more staining in irradiated skin than in nonirradiated skin,
but the difference in staining intensity is consistently greater with
the WT mice than the KO mice (data not shown).
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). There are interesting differences in the expression
patterns of TGF-ß2 in the keratinocytes of irradiated WT and KO skin.
In WT irradiated skin, immunoreactive TGF-ß2 is confined to the outer
layers of the epidermis (stratum granulosum), whereas in irradiated KO
skin, staining is also apparent in the stratum spinosum (Figure 3, G and H)
. The staining pattern in the irradiated KO is similar to that
seen in nonirradiated WT and KO skin where there is relatively uniform
staining of the epidermis with anti-TGF-ß2 (data not shown). In
contrast to the increased epidermal staining for TGF-ß1 after
irradiation, levels of immunoreactive TGF-ß3 in the epidermis
decrease in both irradiated WT and KO mice as compared to nonirradiated
skin (data not shown).
We also compared the immunohistochemical staining of TGF-ß type I
(TßRI) and II (TßRII) receptors in skin before and after
irradiation to determine which cells were capable of responding to
TGF-ß. In both WT and KO nonirradiated skin there is light uniform
staining for TßRI in the epidermis with darker staining of dermal
cells and hair follicles. This pattern does not change after
irradiation of either genotype (data not shown). There is, however, a
dramatic change in TßRII expression after irradiation (Figure 4)
. Although normal skin of both WT and
KO mice show strong expression of TßRII in the epidermis, hair
follicles, and many dermal cells (Figure 4, A and B)
, most staining in
the epidermis and hair follicles is lost 6 weeks after irradiation,
even though staining remains high in the dermis (Figure 4, C and D)
.
This pattern of loss of TßRII is observed in both genotypes.
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To examine whether the observed differences in the histology
of the skin from WT and KO mice after irradiation might be based, in
part, on a genotypic difference in survival of cells after exposure to
irradiation, we isolated primary keratinocytes and dermal fibroblasts
from 1- to 3-day-old WT and KO mice and exposed the cells to various
doses of -irradiation. There is no significant difference in
survival between WT and KO keratinocytes (Figure 5A)
and fibroblasts (Figure 5B)
when
evaluated 2 and 5 days after irradiation, respectively. We also
examined the survival of WT and KO mice after whole-body irradiation.
There is no significant difference in survival of WT, Het, and KO mice
30 days after whole-body irradiation at doses of 6, 7, 8, or 10 Gy. A
dose of 8 Gy is lethal for all genotypes (data not shown), suggesting
that the loss of Smad3 does not afford protection from bone marrow
toxicity.
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Exposure to ionizing radiation has been reported to increase production of both total and active48 TGF-ß within several hours after exposure and persisting for months.31,33 Similar to the mechanisms we have proposed to contribute to the reduced expression of TGF-ß1 in granulation tissue of KO mice after wounding,30 we suggest that the impaired autoinduction of TGF-ß1 in Smad3-null macrophages and fibroblasts,25,30 most likely accounts for a significant portion of the difference in expression of TGF-ß1 after irradiation. Although Smad2-null mice are not viable, comparison of wound healing in Smad2 and Smad3 heterozygous mice showed that the reduced influx of inflammatory cells and reduced accumulation of matrix proteins were Smad3-dependent and not Smad2-dependent.30 Studies using mouse embryo fibroblasts null for either Smad2 or Smad3 also showed that autoinduction of TGF-ß1 selectively requires Smad3 and not Smad2.25 Together, these results suggest that selective ablation of Smad3 may both eliminate or reduce amplification and sustained expression of TGF-ß and chemotactic responses to TGF-ß, thus having particular importance for fibrotic processes.
Two hypotheses can be put forth to explain the phenotypic differences observed in the WT and KO mice after irradiation. On the one hand, loss of Smad3 may alter early signaling events induced by irradiation resulting in different outcomes at later time points. Alternatively, the initial signaling events after irradiation may be independent of the Smad3 status, but the inability of the Smad3-null mice to chronically express and amplify TGF-ß1 in response to these signals may subsequently lead to a resolution of the tissue reaction in KO mice and ultimately to reduced fibrosis. We are currently analyzing samples at a variety of time points after irradiation to help differentiate between these two possibilities, but the data on hand, support the second hypothesis.
Ionizing radiation activates a complex array of cellular signal
transduction pathways including AP-1, nuclear factor-
B,
mitogen-activated protein kinases, phosphatidyl inositol-3-phosphate
kinase (PI-3 kinase), as well as DNA-dependent protein kinase
(DNA-PK), poly(ADP ribose) polymerase (PARP), and ATM
(ataxia-telangiectasia-mutated) that act upstream of
p53.49-52
To address the first hypothesis, we attempted
to determine whether any of these signaling pathways is altered in
Smad3 WT and KO skin at early times after irradiation. Our initial
in vitro analyses of irradiated WT and KO primary cultures
of keratinocytes and dermal fibroblasts show no difference in
activation of AP-1, nuclear factor-B, mitogen-activated protein
kinases, or p53 phosphorylation (K. Flanders and M. Fujii, unpublished
observations). However, study of these cell types in isolation may not
appropriately model early signaling events as occur in vivo.
Additionally, the activation of a multiplicity of transcription factors
and signaling pathways by radiation leads to the induction of a variety
of growth factors and cytokines including TGF-ß, tumor necrosis
factor-
, interleukin-1, fibroblast growth factor, and
platelet-derived growth factor that could then secondarily affect
signaling.53
We have shown that Smad3 KO skin produces
less TGF-ß after irradiation and that KO cells respond differently to
TGF-ß than do WT cells.30
However, the cross-talk
between the Smad and mitogen-activated protein kinase signaling
pathways also raises the possibility that Smad3 WT and KO cells may
differ in their production of or response to cytokines other than
TGF-ß. To address this, we are currently characterizing the cytokine
profile of WT and KO skin after irradiation.
Immunohistochemical staining shows a dramatic decrease in the expression of TßRII in the epidermis in both WT and KO mice after irradiation, while its expression remains high in dermal cells. These effects are selective for the ligand-binding TßRII as no changes after irradiation were observed for TßRI. Recently, it has been reported that ultraviolet radiation has similar effects on mink lung epithelial cells in vitro, resulting in down-regulation of TßRII without altering levels of TßRI protein.54 Selective loss of TßRII expression has also been reported in a number of pathological conditions including carcinomas55,56 and in atherosclerotic lesions.57 In tumor cells, where effects of receptor loss have been studied extensively, it is clear that except in the rare cases of biallelic mutation that result in complete loss of TGF-ß signaling, epigenetic mechanisms contributing to receptor loss likely alter, rather than completely ablate, TGF-ß signaling.55 Typically, such receptor down-regulation results in loss of the growth inhibitory effects of TGF-ß while still allowing for signaling to other target genes that may have a lower signaling threshold.58 In the case of loss of TßRII in epidermal keratinocytes after irradiation, it could be argued that this would ablate the potent growth inhibitory effects of TGF-ß normally seen in these cells59,60 and in that manner contribute to the epidermal hypertrophy seen in the WT epidermis. However, this same reasoning would then also predict epidermal hypertrophy in the KO skin, because these keratinocytes have lost a key signaling intermediate, Smad3, in addition to the receptor loss. The significantly smaller acanthotic response in the KO epidermis instead suggests either that loss of Smad3 has altered other responses to TGF-ß that may remain intact in the WT cells, or that KO keratinocytes may also have an altered response pattern to factors other than TGF-ß1, including mitogens such as keratinocyte growth factor. The control of keratinocyte proliferation in vivo is complex and has been shown to be dependent on a double-paracrine interaction with fibroblasts in which interleukin-1 secreted by keratinocytes induces fibroblasts to secrete keratinocyte growth factor that then promotes keratinocyte proliferation.61,62 Deletion of Smad3 could alter this signaling loop, or, alternatively, TGF-ß may affect keratinocyte growth factor expression by fibroblasts. An example of this exists in limbal fibroblasts where TGF-ß treatment markedly up-regulates protein levels of keratinocyte growth factor despite down-regulation of its transcript levels.63
The high levels of receptor expression in the dermis of both WT and KO skin after irradiation suggest that fibroblasts and inflammatory cells remain responsive to TGF-ß signals and that the striking difference in the numbers of inflammatory cells present in the WT and KO dermis 6 weeks after irradiation likely reflects effects of loss of Smad3 and its role in recruitment of these cells. TGF-ß is chemotactic for mast cells,9,10 monocytes,6 and neutrophils.7,8 Of these, loss of Smad3 is known to impair the chemotactic response of both neutrophils24 and macrophages to TGF-ß30 and the likelihood is that it may impair chemotactic effects of TGF-ß on the other cells as well. Our results suggest, for example, that Smad3 may also function in the TGF-ß-mediated chemotaxis of mast cells, because a twofold increase in these cells was found in the dermis of WT mice compared to KO mice after irradiation. Neutrophils, previously shown to require Smad3 for chemotaxis to TGF-ß, show a threefold difference. In contrast, we observed a similar radiation-dependent fold-increase in the number of macrophages in WT and KO skin (7.7-fold and 6.5-fold, respectively), suggesting that signals activated by irradiation other than TGF-ß are involved in recruitment of these cells. Additional studies will be needed to determine whether the decreased numbers of inflammatory cells in KO animals result primarily from impaired chemotaxis to TGF-ß because of loss of Smad3 or whether the lesser amounts of TGF-ß in the KO skin also contribute to this effect.
Although we have not directly quantitated collagen levels, we
hypothesize that KO mice will, at later times, exhibit a significantly
reduced fibrotic response compared to WT mice after irradiation because
the dermis of KO mice contains one-half the number of myofibroblasts as
determined by -smooth muscle actin staining and because those
myofibroblasts are exposed to reduced levels of TGF-ß. The fibrotic
response is characterized by the transdifferentiation of fibroblasts
into myofibroblasts that acquire contractile properties and secrete
matrix proteins. This conversion, which is transitory in normal wound
healing, does not resolve in chronic tissue fibrosis. Because TGF-ß
has been implicated in the fibroblast to myofibroblast
transition,44-46
the reduced numbers of myofibroblasts
seen in the KO skin may be a result of the lower levels of TGF-ß
expressed in these animals or alternatively, the transition may be
directly dependent on Smad3. Growth inhibitory functions of TGF-ß
have been reported to be lost as fibroblasts differentiate to
myofibroblasts, despite retention of receptor expression levels and
unchanged expression levels of Smad proteins.64
Experiments using primary cultures of pig myofibroblasts suggest that
there is a specific defect in nuclear translocation of Smad3 in these
cells underlying their escape from TGF-ß growth
inhibition.65
Although the complex milieu of cytokines
that the cells are exposed to in vivo makes it difficult to
compare our results directly with these in vitro studies,
our data showing fewer myofibroblasts in KO mice after irradiation
suggest that Smad3 may participate in both the recruitment of
fibroblasts to the site of injury and possibly also the differentiation
of fibroblasts to myofibroblasts. In addition to the reduced numbers of
myofibroblasts in KO dermis, the dependence of TGF-ß-induced
synthesis of collagens 1, 3, 5, and 6 on Smad3, also is strongly
suggestive that loss of Smad3 will be protective against radiation
fibrosis.29,66
Future studies will be aimed at evaluating
such fibrotic endpoints directly, using later time points acquired from
surviving members of irradiated cohorts.
It is not surprising that loss of Smad3 offers no protection against systemic effects of radiation caused by depletion of bone marrow progenitors or against radiation-induced death of cells in vitro. Rather, our prediction is that Smad3 effects will be seen in inflammatory and fibrotic components of the response of tissue to irradiation. Whether our results can be extended to other sites such as lung and intestine where fibrosis is common after radiation of patients, is not yet known. Regardless, our results provide some optimism that the ability to protect normal tissue during radiotherapy by interfering with specific signaling pathways could allow dose escalation for more effective tumor kill while decreasing the inflammatory response and the fibrotic sequelae that adversely affect a patient’s quality of life. Clearly the development of a selective Smad3 inhibitor could have important applications in radiation therapy.
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Acknowledgements |
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Footnotes |
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Accepted for publication December 13, 2001.
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