Current Eye Research
Volume 11 number 7 1992, 691 -696
Transforming growth factor-@modulates effects of epidermal growth factor on corneal epithelial cells ~
~
~
~~~~
~
Hiroshi Mishima. Masatsugu Nakamura, Junko Murakami, Teruo Nishida and Toshifumi Otori
Curr Eye Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/23/14 For personal use only.
Department of Ophthalmology, Kinki University School of Medicine, 377-2 Ohno-Higashi, Osaka-Sayama City, Osaka 589, Japan
ABSTRACT In order to understand the mechanisms that bring about maintenance and restoration of the integrity of corneal epithelium, we investigated independent and combined effects of transforming growth factor-p (TGF-p) and epidermal growth factor (EGF) on rabbit corneal epithelial cells in cell and organ culture. Specifically, we determined whether incubation with these factors influenced 1) cellular proliferation, 2) ability of cells to attach to a fibronectin matrix, and 3) the rate of epithelial migration over corneal stroma. Incubation with TGF-p caused a dose-related decrease in the incorporation of 3H-thymidine by the epithelial cells. EGF increased 3H-thymidine incorporation, but this effect was antagonized by the addition of TGF-p into the incubation medium. Incubation with EGF increased the numbers of cells that attached to a fibronectin matrix. TGF-p itself did not affect the number of attached cells but, again, it antagonized the stimulatory effect of EGF. Similarly, when corneal blocks were cultured with EGF, epithelial migration increased in a dose-related manner. TGF-p itself did not affect epithelial migration a t any of the concentrations tested (0.1-10 ng/ml), but it antagonized EGF-stimulated epithelial migration. These findings suggest that the proliferation and the migration of corneal epithelial cells are regulated by different mechanisms, and that TGF-p serves as a modulator of the effects of EGF.
important t o understand the actions and interrelating
effects of these substances which determine t h e physiology and pathology of the corneal epithelium. Epidermal growth factor (EGF), a polypeptide with 53 amino acids, stimulates both the growth of skin epidermal cells and keratinization (1). In the cornea, it stimulates epithelial cell proliferation (2,3) and reepithelialization (4-6). EGF also stimulates fibronectin synthesis by corneal cells (7). We have recently showed that its stimulatory effect on corneal epithelial migration is mediated by a fibronectindependent mechanism ( 8 ) . EGF is present in tears of both normal and inflamed eyes (9-12). Thus the corneal epithelium is continuously exposed to EGF. Apparently, some factors normally counteract the actions of EGF, preventing excessive cellular proliferation. Transforming growth factor p (TGF-p), a 25kDa polypeptide, is thought to be a modulator of inflammation and wound healing. I t has been reported to stimulate healing of wounded skin (13,141, but it has also been reported to be a potent inhibitor of the
INTRODUCTION The epithelium must maintain its normal
proliferation of epithelial and endothelial cells (15-1 7).
continuous integrity in order to protect the internal environment of the body. Corneal epithelium must
Furthermore, TGF-p has been reported to modulate the
have a well-organized, layered structure and smooth surface to ensure good vision. Such a structure
actions of cytokines and of other growth factors such as EGF, fibroblast growth factor and insulin-like growth factor (18-20). Using an immunohistochemical
requires the well-coordinated regulation of cellular proliferation and migration and of intercellular
technique, the presence of TGF-p in the corneal fibroblasts and macrophages after epithelial wounding
adhesion and attachment to extracellular matrix
was reported (21). These findings indicate that TGF-p participates in the system of growth factors and
proteins. Specific growth factors, cytokines, and extracellular matrix proteins are known to participate
cytokines that regulate the maintenance and healing of
in this process, but their precise roles and
the corneal epithelium.
relationships to each other remain unclear.
I t is
In order to better understand the mechanisms of
Received on March 16, 1992; accepted on June 9, 1992
@ Oxford University Press
691
Current Eye Research corneal epithelial wound healing, we studied the independent and combined actions of TGF-p and EGF on corneal epithelial cells. Using cultured cells, we examined the effects of TGF-p and EGF both on cellular proliferatlon (i.e., DNA synthesis) by measuring 'H-thymidine incorporation and on the expression of fibronectin receptor (integrlns) by measuring the effect of prior incubation with EGF and/or TGF-p on cellular
Curr Eye Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/23/14 For personal use only.
attachment to a fibronectin matrix. Using blocks of cultured corneas, we also examined the effects of
3H-TdR incorporation Corneal epithelial cells were cultured on 96multiwell culture plates (5x104 cells/well) for 4 days. The culture medium was then changed to TC-199 containing 1%FBS (low-serum medium) to which one of the followings was added:
1) TGF-p a t the
concentrations of 0.3, 1, 3, or 10 ng/ml; 2 ) EGF a t the concentrations of 0.1, 1, or 10 ng/ml; or 3) EGF (same concentrations) plus TGF-p (10 ng/ml). No growth factor was added to control cultures.
Twenty-
TGF-p and EGF on epithelial migration.
four hours later, 3H-TdR (3.7 KBq/well, 37 KBq/ml) was added. After 24 hours, the cells were harvested
MATERIALS AND METHODS Albino male rabbits were purchased from
and radioactivity was measured with a scintillation
Hokusetsu Sangyo Co. (Settsu, Osaka, Japan). Care
counter. Data were expressed as mean ( 2 SEMI cpm/well in a triplicated or quadruplicated assay.
and treatment of animals adhered to the Guiding Principles in the Care and Use of Animals (DHEW Publication, NIH 86-23). TC-199 culture medium,
Attachment
trypsin solution (0.25%) and EDTA solutlon (0.02%)
epithelial cells were cultured with 15% FBS in the TC199 culture medium for 3 days. The culture media were then changed to fresh low-serum media containing
were obtained from the Research Foundation for Microbial Diseases of Osaka University (Suita, Osaka, Japan); fetal bovine serum (FBS) was from Flow
fibronectin matrix
The attachment assay was performed as described previously (22) with slight modification. Corneal
TGF-p (10 ng/ml), EGF (10 ng/ml), or both.
Control
Laboratories (North Ryde, Australia); dispase (300,000 units/g) was from Sanko Junyaku (Tokyo, Japan);
media contained no growth factor.
Bovine serum albumin (BSA) was from Nakarai Tesque Inc. (Kyoto, Japan); human recombinant EGF and human recombinant TGF-p were from Earth
plates (1,000 cells/well) precoated with BSA (control plates) or fibronectin (10 pg/ml) plus BSA as described previously (22). and incubated for 45
Pharmaceutical Co. (Akoh, Hyogo, Japan); human
After 24 hours, the cells were harvested, plated on 96-multlwell plastic
plasma fibronectin was from New York Blood Center
minutes. After the cells were fixed and stained, the numbers of attached cells were counted under a phase
(New York, NY). Tissue culture flasks and multiwell tissue culture plates (96 wells and 24 wells) were from Costar (Cambridge, MA). [meth~l-~HI-Thymidine
contrast microscope. Data were expressed as mean SEMI of the number of attached cells/well in a triplicated assay.
deoxyribose (3H-TdR, TRK 686, 3.15 TBq/mmol) was obtained from Amersham Japan (Tokyo, Japan).
Epithelial migration
Cell culture --
culture of the rabbit cornea was measured by a
Corneal epithelial cells were prepared and cultured as described previously (22). In brief, rabbit
previously described method (23). Briefly, rabbits were killed by intravenous injection of pentobarbital.
corneas were excised, the endothelial layers were removed, and the corneas were incubated with dispase
Corneas were excised and cut into the small blocks
( 2 mg/ml in TC-199 medium) for 1 hour. The epithelial layer was harvested and further incubated with trypsin (0.125%) and EDTA (0.01%)to make
24-multiwell culture plates and incubated for 24 hours
single cell suspensions. The cells were plated in culture flasks and cultured with 15%FBS in the TC199 medium.
same concentrations plus EGF at 10 ng/ml; 3) EGF a t the concentrations of 0.1, 1, or 10 ng/rnl; 4) EGF at the same concentrations plus TGF-p a t 10 ng/ml.
692
(5
The length of the path of epithelial migration in
(approximately 2 x 4 mm).
The blocks were plated in
in the TC-199 medium containing: 1) TGF-p a t the concentrations of 0.1, 1, or 10 ng/ml; 2) TGF-p a t the
Current Eye Research Control blocks were incubated without growth factors.
RESULTS
Thin sections were cut, stained by hematoxylin-eosin, and photomicrographed. The length of the path of
Effects of TGF-p and EGF on 'H-TdR incorporation are represented in Figures 1 and 2. Addition of TGF-p alone to the culture medium decreased 'H-TdR incorporation by corneal epithelial
epithelial migration over the corneal stroma was measured on the photomicrographs. Data were
cells in a dose-dependent manner (Figure 1). A t the concentration of 3 or 10 ng/ml of TGF-p, this
expressed as mean (f SEM) pm of 6 measurements. Statistics Statistical significance was determined by
decrease was statistically significant (P
Volume 11 number 7 1992, 691 -696
Transforming growth factor-@modulates effects of epidermal growth factor on corneal epithelial cells ~
~
~
~~~~
~
Hiroshi Mishima. Masatsugu Nakamura, Junko Murakami, Teruo Nishida and Toshifumi Otori
Curr Eye Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/23/14 For personal use only.
Department of Ophthalmology, Kinki University School of Medicine, 377-2 Ohno-Higashi, Osaka-Sayama City, Osaka 589, Japan
ABSTRACT In order to understand the mechanisms that bring about maintenance and restoration of the integrity of corneal epithelium, we investigated independent and combined effects of transforming growth factor-p (TGF-p) and epidermal growth factor (EGF) on rabbit corneal epithelial cells in cell and organ culture. Specifically, we determined whether incubation with these factors influenced 1) cellular proliferation, 2) ability of cells to attach to a fibronectin matrix, and 3) the rate of epithelial migration over corneal stroma. Incubation with TGF-p caused a dose-related decrease in the incorporation of 3H-thymidine by the epithelial cells. EGF increased 3H-thymidine incorporation, but this effect was antagonized by the addition of TGF-p into the incubation medium. Incubation with EGF increased the numbers of cells that attached to a fibronectin matrix. TGF-p itself did not affect the number of attached cells but, again, it antagonized the stimulatory effect of EGF. Similarly, when corneal blocks were cultured with EGF, epithelial migration increased in a dose-related manner. TGF-p itself did not affect epithelial migration a t any of the concentrations tested (0.1-10 ng/ml), but it antagonized EGF-stimulated epithelial migration. These findings suggest that the proliferation and the migration of corneal epithelial cells are regulated by different mechanisms, and that TGF-p serves as a modulator of the effects of EGF.
important t o understand the actions and interrelating
effects of these substances which determine t h e physiology and pathology of the corneal epithelium. Epidermal growth factor (EGF), a polypeptide with 53 amino acids, stimulates both the growth of skin epidermal cells and keratinization (1). In the cornea, it stimulates epithelial cell proliferation (2,3) and reepithelialization (4-6). EGF also stimulates fibronectin synthesis by corneal cells (7). We have recently showed that its stimulatory effect on corneal epithelial migration is mediated by a fibronectindependent mechanism ( 8 ) . EGF is present in tears of both normal and inflamed eyes (9-12). Thus the corneal epithelium is continuously exposed to EGF. Apparently, some factors normally counteract the actions of EGF, preventing excessive cellular proliferation. Transforming growth factor p (TGF-p), a 25kDa polypeptide, is thought to be a modulator of inflammation and wound healing. I t has been reported to stimulate healing of wounded skin (13,141, but it has also been reported to be a potent inhibitor of the
INTRODUCTION The epithelium must maintain its normal
proliferation of epithelial and endothelial cells (15-1 7).
continuous integrity in order to protect the internal environment of the body. Corneal epithelium must
Furthermore, TGF-p has been reported to modulate the
have a well-organized, layered structure and smooth surface to ensure good vision. Such a structure
actions of cytokines and of other growth factors such as EGF, fibroblast growth factor and insulin-like growth factor (18-20). Using an immunohistochemical
requires the well-coordinated regulation of cellular proliferation and migration and of intercellular
technique, the presence of TGF-p in the corneal fibroblasts and macrophages after epithelial wounding
adhesion and attachment to extracellular matrix
was reported (21). These findings indicate that TGF-p participates in the system of growth factors and
proteins. Specific growth factors, cytokines, and extracellular matrix proteins are known to participate
cytokines that regulate the maintenance and healing of
in this process, but their precise roles and
the corneal epithelium.
relationships to each other remain unclear.
I t is
In order to better understand the mechanisms of
Received on March 16, 1992; accepted on June 9, 1992
@ Oxford University Press
691
Current Eye Research corneal epithelial wound healing, we studied the independent and combined actions of TGF-p and EGF on corneal epithelial cells. Using cultured cells, we examined the effects of TGF-p and EGF both on cellular proliferatlon (i.e., DNA synthesis) by measuring 'H-thymidine incorporation and on the expression of fibronectin receptor (integrlns) by measuring the effect of prior incubation with EGF and/or TGF-p on cellular
Curr Eye Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/23/14 For personal use only.
attachment to a fibronectin matrix. Using blocks of cultured corneas, we also examined the effects of
3H-TdR incorporation Corneal epithelial cells were cultured on 96multiwell culture plates (5x104 cells/well) for 4 days. The culture medium was then changed to TC-199 containing 1%FBS (low-serum medium) to which one of the followings was added:
1) TGF-p a t the
concentrations of 0.3, 1, 3, or 10 ng/ml; 2 ) EGF a t the concentrations of 0.1, 1, or 10 ng/ml; or 3) EGF (same concentrations) plus TGF-p (10 ng/ml). No growth factor was added to control cultures.
Twenty-
TGF-p and EGF on epithelial migration.
four hours later, 3H-TdR (3.7 KBq/well, 37 KBq/ml) was added. After 24 hours, the cells were harvested
MATERIALS AND METHODS Albino male rabbits were purchased from
and radioactivity was measured with a scintillation
Hokusetsu Sangyo Co. (Settsu, Osaka, Japan). Care
counter. Data were expressed as mean ( 2 SEMI cpm/well in a triplicated or quadruplicated assay.
and treatment of animals adhered to the Guiding Principles in the Care and Use of Animals (DHEW Publication, NIH 86-23). TC-199 culture medium,
Attachment
trypsin solution (0.25%) and EDTA solutlon (0.02%)
epithelial cells were cultured with 15% FBS in the TC199 culture medium for 3 days. The culture media were then changed to fresh low-serum media containing
were obtained from the Research Foundation for Microbial Diseases of Osaka University (Suita, Osaka, Japan); fetal bovine serum (FBS) was from Flow
fibronectin matrix
The attachment assay was performed as described previously (22) with slight modification. Corneal
TGF-p (10 ng/ml), EGF (10 ng/ml), or both.
Control
Laboratories (North Ryde, Australia); dispase (300,000 units/g) was from Sanko Junyaku (Tokyo, Japan);
media contained no growth factor.
Bovine serum albumin (BSA) was from Nakarai Tesque Inc. (Kyoto, Japan); human recombinant EGF and human recombinant TGF-p were from Earth
plates (1,000 cells/well) precoated with BSA (control plates) or fibronectin (10 pg/ml) plus BSA as described previously (22). and incubated for 45
Pharmaceutical Co. (Akoh, Hyogo, Japan); human
After 24 hours, the cells were harvested, plated on 96-multlwell plastic
plasma fibronectin was from New York Blood Center
minutes. After the cells were fixed and stained, the numbers of attached cells were counted under a phase
(New York, NY). Tissue culture flasks and multiwell tissue culture plates (96 wells and 24 wells) were from Costar (Cambridge, MA). [meth~l-~HI-Thymidine
contrast microscope. Data were expressed as mean SEMI of the number of attached cells/well in a triplicated assay.
deoxyribose (3H-TdR, TRK 686, 3.15 TBq/mmol) was obtained from Amersham Japan (Tokyo, Japan).
Epithelial migration
Cell culture --
culture of the rabbit cornea was measured by a
Corneal epithelial cells were prepared and cultured as described previously (22). In brief, rabbit
previously described method (23). Briefly, rabbits were killed by intravenous injection of pentobarbital.
corneas were excised, the endothelial layers were removed, and the corneas were incubated with dispase
Corneas were excised and cut into the small blocks
( 2 mg/ml in TC-199 medium) for 1 hour. The epithelial layer was harvested and further incubated with trypsin (0.125%) and EDTA (0.01%)to make
24-multiwell culture plates and incubated for 24 hours
single cell suspensions. The cells were plated in culture flasks and cultured with 15%FBS in the TC199 medium.
same concentrations plus EGF at 10 ng/ml; 3) EGF a t the concentrations of 0.1, 1, or 10 ng/rnl; 4) EGF at the same concentrations plus TGF-p a t 10 ng/ml.
692
(5
The length of the path of epithelial migration in
(approximately 2 x 4 mm).
The blocks were plated in
in the TC-199 medium containing: 1) TGF-p a t the concentrations of 0.1, 1, or 10 ng/ml; 2) TGF-p a t the
Current Eye Research Control blocks were incubated without growth factors.
RESULTS
Thin sections were cut, stained by hematoxylin-eosin, and photomicrographed. The length of the path of
Effects of TGF-p and EGF on 'H-TdR incorporation are represented in Figures 1 and 2. Addition of TGF-p alone to the culture medium decreased 'H-TdR incorporation by corneal epithelial
epithelial migration over the corneal stroma was measured on the photomicrographs. Data were
cells in a dose-dependent manner (Figure 1). A t the concentration of 3 or 10 ng/ml of TGF-p, this
expressed as mean (f SEM) pm of 6 measurements. Statistics Statistical significance was determined by
decrease was statistically significant (P
