TGF-beta 3, but Not TGF-beta 1, Protects Keratinocytes against 12-O-Tetradecanoylphorbol-13-acetate-induced Cell Death in Vitro and in Vivo*

Jie LiDagger §, Kerstin FoitzikDagger , Enzo CalauttiDagger , Howard BadenDagger , Tom Doetschman, and G. Paolo DottoDagger parallel

From the Dagger  Cutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts 02129 and Department of Molecular Genetics Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0524

    ABSTRACT
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Abstract
Introduction
References

We have examined the role that individual TGF-beta isoforms, and in particular TGF-beta 3, play in control of epidermal homeostasis. Mice with a knockout mutation of the TGF-beta 3 gene die a few hours after birth. A full-thickness skin grafting approach was used to investigate the postnatal development and homeostatic control of the skin of these mice. Grafted skin of mice with a disruption of the TGF-beta 3 gene developed similarly to grafts of wild type and TGF-beta 1 knockout animals. However, a strikingly different response was observed after acute treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). When exposed to TPA, the grafted skin of wild type and TGF-beta 1 knockout mice underwent a hyperplastic response similar to that of normal mouse skin. In marked contrast, TPA treatment of TGF-beta 3 knockout grafts induced widespread areas of keratinocyte cell death. Analysis of cultured keratinocytes treated with purified TGF-beta isoforms revealed that TGF-beta 3 plays a direct and specific function in protecting keratinocytes against TPA-induced cell death. The protective function of TGF-beta 3 on TPA-induced cell death was not because of general suppression of the signaling pathways triggered by this agent, as ERK1/2 activation occurred to a similar if not greater extent in TGF-beta 3-treated versus control keratinocytes. Instead, TGF-beta 3 treatment led to a significant reduction in TPA-induced c-Jun N-terminal kinase activity, which was associated and possibly explained by specific counteracting effects of TGF-beta 3 on TPA-induced disruption of keratinocyte focal adhesions.

    INTRODUCTION
Top
Abstract
Introduction
References

Homeostasis of self renewing epithelia such as the epidermis is determined by a complex interplay of signals, resulting from cell-cell and cell-matrix interactions as well as diffusible factors acting on cells of either the same or different type. The TGF-beta family of diffusible factors is thought to play a key role in control of epithelial homeostasis. Some well known functions of TGF-beta s in epithelial tissues include growth inhibition, promotion of cell adhesion and tissue reorganization, suppression and, at later stages, promotion of tumor development (1). Three TGF-beta isoforms exist that share a high degree of homology (~80%) in their processed active regions. All three isoforms bind to the same cellular receptors, although with different affinities, and elicit similar biological responses on most cultured cells. However, expression of the three TGF-beta s is differentially regulated in vivo in different cell types and at various developmental stages, suggesting that the three isoforms play distinct biological roles (2). This conclusion is consistent with the fact that mice with disruptions of each of the three TGF-beta genes have little or no overlapping phenotype. TGF-beta 1 knockout mice die a few weeks after birth of a wasting syndrome accompanied by a multifocal inflammatory reaction (3, 4). Mice with a knockout mutation of the TGF-beta 3 gene die a few hours after birth because of cleft palate (5, 6). Mice with a disruption of the TGF-beta 2 gene die also at birth, because of a relatively large number of developmental abnormalities affecting the cardiovascular and musculoskeletal systems as well as other organs (7).

In the skin, elevated TGF-beta 1 expression has been found in keratinocytes of the basal epidermal layer and in cultured keratinocytes under basal proliferating conditions, suggesting that this factor is involved in a negative feedback growth regulatory loop (8, 9). TGF-beta 2 expression is increased in keratinocytes upon differentiation (10), implicating this isoform as a possible mediator of differentiation-induced growth arrest.

Expression of TGF-beta 3 mRNA is limited, at least in culture, to cells of mesenchymal origin, including dermal fibroblasts, whereas it is not found in keratinocytes (11, 12). This factor is likely to diffuse from the dermis to the overlying epidermis (9) and may be an important mediator of dermal-epidermal interactions, including those involved in the wound-healing reaction (13, 14) as well as in negative control of keratinocyte tumor development (12).

Skin homeostasis results from a delicate balance between keratinocyte growth, differentiation, and apoptosis. Such a balance is perturbed upon exposure to tumor promoting and/or inflammatory agents, UV light, and wounding. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA)1 is a potent skin tumor promoter. In vivo, acute treatment with TPA leads to epidermal hyperplasia, possibly through mechanisms such as blockage of gap-junctional intercellular communication (15) and/or differential modulation of specific TGF-beta isoforms (9, 16). In vitro, acute TPA treatment has the opposite effect of inducing keratinocyte detachment, growth arrest, and differentiation (Ref. 17 and references therein). The discrepancy between the in vivo and in vitro effects has never been explained.

The present study was undertaken to establish the role that individual TGF-beta s, and in particular TGF-beta 3, play in control of epidermal homeostasis. As mentioned, mice with a disruption of the TGF-beta 3 gene die a few hours after birth (5, 6). A full-thickness skin grafting approach was used to investigate the postnatal development and homeostatic control of the skin of these mice, in comparison with similarly grafted skin from wild type and TGF-beta 1 knockout animals. These studies suggest that TGF-beta 3 may not be essential for normal skin maturation. Instead, the in vivo findings, together with analysis of cultured keratinocytes treated with purified TGF-beta isoforms, indicate that TGF-beta 3 plays a specific and direct function in protecting keratinocytes against the death-inducing effects of agents such as TPA.

    EXPERIMENTAL PROCEDURES

Skin Grafting Experiments-- Homozygous TGF-beta 3 knockout mice die at birth and can be rapidly cannibalized by the mothers. For this reason, experiments were synchronized so as to have sufficient numbers of time-mated heterozygous pregnant females to use as source of pups immediately before delivery (18.5 days of pregnancy). The genotype of pups was determined by polymerase chain reaction analysis of tail DNA with oligonucleotide primers specific for the wild-type and disrupted TGF-beta alleles (6). Skins from wild type and homozygous knockout littermates were used for full-thickness grafting onto nude mice. The procedure involved a 10 × 10-mm excision of the host skin, up to but not including the panniculus carnosus and placement of a similar size fragment of newborn mouse skin onto the area of excision. The grafted skin was covered with a Vaseline gauze and a silicon transplantation chamber on top for protection. Gauze and chamber were removed 6 days after grafting. Grafts were analyzed at 1 month after grafting, under basal conditions and after treatment with TPA (10-4 M in acetone, Sigma) or acetone vehicle alone. In each case, the grafted tissue was excised, half was frozen, and the other half was fixed in 10% neutral-buffered formalin before histological and immunohistochemical analysis. Terminal deoxynucleotidyltransferase-mediated dUTP biotin nick end-labeling (TUNEL) assays of frozen sections were performed as described below for cultured cells. Immunohistochemistry for TGF-beta 3 expression was performed by incubating frozen sections of wild type and knockout grafted skins with antibodies against TGF-beta 3 (Santa Cruz) in the presence or the absence of a specific neutralizing peptide followed by incubation with biotinylated anti-rabbit secondary antibodies (Vector, Burlingame). Fast RED (Sigma) was used as substrate for the alkaline phosphatase reaction.

Cell Culture and Viability Assays-- Primary keratinocytes were prepared from wild type newborn mice (SENCAR) and grown at 34 °C and 8% CO2 in medium at low calcium concentrations as described previously (18). Confluent cultures were tested 5 days after plating. Cells were either untreated or treated with the TGF-beta 1 or TGF-beta 3 isoforms (R&D Systems) at 3 ng/ml for the indicated amounts of time followed by exposure to TPA (in Me2SO stock solutions; 100 ng/ml final concentration) or Me2SO vehicle alone. Cell viability was determined by the calcein-AM/propidium iodide staining method (19). Briefly, culture medium was replaced with 1 µM calcein-AM (Molecular Probes) and 5 µg/ml propidium iodide in serum-free low calcium medium for 20 min at 34 °C. Cultures were examined immediately without fixation using a Zeiss Axiophot fluorescence microscope. Green and red fluorescence were simultaneously recorded using a computer-digitalized equipment. In each case, 4-5 different fields were examined, and a minimum of 800 cells were counted to determine the fraction of calcein-AM (green) versus propidium iodide (red) positive cells.

TUNEL Assays-- Primary keratinocytes that were either untreated or treated with specific TGF-beta isoforms and/or TPA were trypsinized, centrifuged, and brought into suspension into serum-supplemented medium, counted, and recentrifuged in a cytospin apparatus onto gelatin-coated glass slides. In other experiments, cells were directly grown on tissue culture slides (Labtec) and treated under these conditions. Cytospin preparations or keratinocytes directly grown on slides were fixed in 10% neutral-buffered formalin for 10 h at room temperature, air dried for 1 h, and then permeabilized with 20 µg/ml proteinase K for 5-10 h at room temperature. The terminal deoxynucleotidyltransferase-mediated dUTP biotin nick end-labeling assay (TUNEL) was performed using the TACS terminal deoxynucleotidyltransferase in situ apoptosis detection kit (Trevigen) using 3,3'-diaminobenzidine tetrahydrochloride or TUNEL blue labeling for detection according to the manufacturer's instructions.

Immunofluorescence Analysis of Focal Adhesions and Actin Cables-- Mouse primary keratinocytes were plated onto collagen-coated glass coverslips and grown to confluence. Cells that were either untreated or treated with TGF-beta 1, TGF-beta 3, and/or TPA were fixed for 10 h at room temperature with 2% paraformaldehyde in phosphate-buffered saline followed by permeabilization with 0.1% Triton X-100 in phosphate-buffered saline and blocking in 5% goat serum in phosphate-buffered saline. Cells were incubated with anti-vinculin rabbit antiserum (Sigma) for 1 h at room temperature, followed by incubation with fluorescein isothiocyanate-conjugated secondary antibodies (Southern Biotech. Associates) and BODIPY(581-591)-conjugated phalloidin (Molecular Probes). Samples were analyzed by confocal microscopy using a LEICA TCS 4D scanner connected to an inverted LEITZ DM IRB microscope. Images were processed using a TCS-NT software package.

In Vitro Kinase Assays-- Primary keratinocytes in low calcium medium were either untreated or treated with TGFbeta 1 or TGFbeta 3 (3 ng/ml) for 18 h followed by exposure to TPA (100 ng/ml) for different lengths of time. In vitro kinase assays were performed using the stress-activated protein kinase/c-Jun N-terminal kinase (JNK) or p44/42 mitogen-activated protein kinase assay kits (New England BioLabs Inc.), according to the manufacturer's instructions. Briefly, cells were lysed in cell lysis buffer and normalized for protein amounts by the Bradford assay (Bio-Rad). Same amounts of total cell extracts (250 µg of proteins) were incubated with c-Jun (1-89) fusion protein beads to immunoprecipitate the Jun N-terminal kinase or with phospho-specific antibody to p44/42 mitogen-activated protein kinase to selectively immunoprecipitate active mitogen-activated protein kinase (ERK1/2). Kinase reactions were carried out in the presence of cold ATP using either recombinant c-Jun or Elk1 proteins as substrates for the JNK and ERK1/2 kinases, respectively. Samples were analyzed by 12% SDS-polyacrylamide gel electrophoresis and immunoblotting with either phospho-specific c-Jun antibodies to detect c-Jun phosphorylated at Ser-63 or phospho-specific Elk1 antibodies to detect the phosphorylated form of Elk1 at Ser-383. For JNK and ERK1/2 protein level determinations, total cell extracts were analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting with rabbit polyclonal antibodies against JNK and ERK1/2, respectively (Santa Cruz Biotech., Inc.). In all cases, blots were developed with the ECL system (Amersham Pharmacia Biotech), and results were quantified by densitometric scanning.

    RESULTS

1) Normal Maturation of the Skin of TGF-beta 3-deficient Mice-- The neonatal mortality of TGF-beta 3-deficient mice prevents a direct assessment of how this mutation can affect the maturation and homeostasis of skin after birth. To overcome this problem, we used a full-thickness grafting approach of newborn mouse skin onto nude mice in such a way that it was possible to compare the effects of the TGF-beta 3 and -beta 1 mutations under identical experimental settings and for a prolonged period of time. Newborn mouse skin grafted in this manner develops normally as if in the intact animal, with good hair growth becoming detectable by 9-10 days after grafting and complete fur formation by 18-20 days. Within a similar time frame, the multiple epidermal layers characteristic of newborn skin are reduced to the two to three layers of the adult animal. The grafts can be maintained up to 1 year with normal hair production.

The lack of the TGF-beta 3 isoform did not lead to gross alterations in skin maturation and hair formation. Compared with control grafts, the grafted skin of mice with specific TGF-beta 3 or -beta 1 knockout mutations showed good hair growth by 2-3 weeks after grafting (Fig. 1 and data not shown). Within a similar time frame, the stratified multiple squamous epidermal layers characteristic of normal newborn mouse skin were reduced to 2-3 layers of the adult mouse epidermis with normal structure and morphology. Immunohistochemical analysis with antibodies against TGF-beta 3 produced a significantly lower level of positive staining in the grafts of TGF-beta 3 knockout skin relative to surrounding skin and/or grafts of wild type animals (Fig. 1, lower panels). However, even in the TGF-beta 3 knockout grafts, residual specific staining for TGF-beta 3 was detectable, especially at the periphery, consistent with some diffusion of this factor from the neighboring skin.


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Fig. 1.   Full-thickness grafting of TGF-beta 3 knockout skin onto nude mice. A, normal maturation of a skin graft derived from a newborn TGF-beta 3 knockout mouse at 1 month after grafting. B, levels of TGF-beta 3 protein in grafts of TGF-beta 3 knockout versus wild type skin at 1 month after grafting as determined by immunohistochemical analysis. Skin grafts from wild type (w.t.) and TGF-beta 3 knockout mice (beta 3-/-) were stained with anti-TGF-beta 3 antibodies as described under "Experimental Procedures." The wild type skin graft was also stained with the antibodies in the presence of a neutralizing peptide. Bar, 250 µm.

2) Widespread Epidermal Cell Death in the Skin of TGF-beta 3-deficient Mice after Phorbol Ester Treatment-- Previous analysis of knockout mice for other potentially redundant genes has indicated that essential functions of these genes can be unmasked under conditions of stress, where normal homeostatic mechanisms are perturbed or partially abrogated (18, 20). One such condition is acute treatment of mouse skin with the phorbol ester tumor promoter TPA, which results in marked epidermal hyperplasia as well as mild inflammatory reaction by 24-48 h of exposure (Ref. 9 and references therein). When treated with TPA, the grafted epidermis of wild type and TGF-beta 1 knockout mice underwent a hyperplastic response similar to that of normal mouse skin (Fig. 2A). Interestingly, a more pronounced inflammatory infiltrate was detectable in the grafts of TGF-beta 1 knockout skins, consistent with the immunosuppressive function of this specific isoform (3). In marked contrast, TPA treatment of TGF-beta 3 knockout grafts induced widespread areas of keratinocyte cell death (Fig. 2A). Keratinocytes in these areas exhibited mixed features of an apoptotic and necrotic process, with highly pyknotic nuclei and cell swelling (Fig. 2A).


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Fig. 2.   Epidermal hyperplasia of wild type and TGF-beta 1 knockout grafts and widespread cell death in TGF-beta 3 knockout grafts in response to acute TPA treatment. A, full-thickness grafts derived from wild type (w.t.), TGF-beta 1 (beta 1-/-), and TGF-beta 3 (beta 3-/-) mice at 1 month after grafting were treated with TPA (10-4 M in acetone, 200 ml) for 24 or 48 h or with control acetone alone for 48 h (Ctrl). The integrity of the grafted skin was verified in each case at the beginning of the experiment, after shaving. Each condition was tested on duplicate mice with similar results. These findings were confirmed in two other independent experiments. Bar, 180 µm. B, grafts of TGF-beta 3 knockout skin treated with acetone alone or TPA for 48 h and of wild type skin treated with TPA were analyzed by TUNEL assay using immunoperoxidase for detection. Bar, 90 µm.

Apoptotic cells as well as cells undergoing necrosis are characterized by increased nuclear DNA fragmentation, which can be detected by TUNEL assays (21). Most of the pyknotic nuclei in the TPA-treated TGF-beta 3 knockout grafts stained positive by this assay, whereas no TUNEL positivity was detected in the TPA-treated wild type grafts nor in the acetone-treated controls (Fig. 2B).

In addition to cell death in the epidermis, the TPA-treated TGF-beta 3 knockout grafts exhibited signs of dermal necrosis. Many venules were also affected by granulomatous vasculitis with segmented fibrinoid necrosis of the cell walls, angiocentric histiocytosis, and endothelial swelling (data not shown). Capillaries showed evidence of microangiopathy with some vessels occluded by fibrin thrombi. None of these changes were found in similarly treated grafts of wild type or TGF-beta 1 knockout skins.

3) Direct Protective Effects of the TGF-beta 3 Isoform on TPA-induced Cell Death of Cultured Keratinocytes-- The alterations of epidermal cells observed in the TGF-beta 3 knockout grafts after TPA treatment may be explained by a direct protective function of TGF-beta 3 on keratinocytes or, alternatively, to an indirect effect on the dermis. These possibilities were further tested in culture by examining whether exogenous addition of either TGF-beta 3 or TGF-beta 1 can protect primary keratinocytes from the effects of TPA. A significant fraction of growing keratinocytes in culture undergoes nuclear DNA fragmentation, which is likely because of the spontaneous continuous shedding of cells into the medium. TPA treatment of control keratinocytes significantly increased the fraction of cells exhibiting nuclear DNA fragmentation as early as 6-8 h after treatment (Fig. 3A). As previously reported for human keratinocytes (22, 23), TGF-beta 1 treatment was also by itself sufficient to increase the number of TUNEL-positive cells. Interestingly, no such effect was observed with TGF-beta 3 (Fig. 3A). More importantly, pretreatment of keratinocytes with this latter isoform suppressed the increase in TUNEL positivity induced by TPA, whereas no such protective effects were observed after pretreatment with TGF-beta 1 (Fig. 3A).


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Fig. 3.   TPA-induced cell death of cultured keratinocytes and protective effects of the TGF-beta 3 isoform. Primary mouse keratinocytes were either left untreated (Ctl) or treated with TGF-beta 1 or TGF-beta 3 for 18 h before exposure to Me2SO vector alone or TPA (100 ng/ml) for an additional 8 h. A, nuclear DNA fragmentation as assessed by TUNEL assay. Left panel, representative fields of control and TGF-beta 3-treated keratinocytes plus/minus TPA exposure. Right panels, composite result of four independent experiments. B, cell viability as assessed by combined staining with calcein-AM (green, viable) and propidium iodide (red, dead). Right panel, composite result of three independent experiments. In all cases, results were quantified by examining at least 4 independent fields, counting a minimum of 800 cells. Values are expressed as fraction of TUNEL or calcein A-positive (i.e. viable) cells.

Staining of cells with calcein-AM/propidium iodide provides an alternative method to TUNEL assays to measure cell death (19). This method relies on the ability of viable cells to cleave calcein/AM into calcein, generating a green fluorescent signal, whereas dying cells take up propidium iodide into the nucleus and become identifiable by red fluorescence. Experiments using this method confirmed that the death-inducing action of TPA was prevented by pretreatment of keratinocytes with the TGF-beta 3 but not TGF-beta 1 isoforms (Fig. 3B).

4) Disruption of Focal Adhesions and Actin Cables in TPA-treated Keratinocytes and Counteracting Effects of TGF-beta 3-- One of the most important consequences of TPA treatment is detachment of keratinocytes from their substratum (Ref. 18 and references therein), which in turn can trigger programmed cell death (24). The protective function of TGF-beta 3 on TPA-induced cell death may be connected with specific effects on keratinocyte cell attachment. Focal adhesions serve as a bridge between integrin receptors and the actin cytoskeleton and provide an essential adhesive structure of keratinocytes in culture (25). Keratinocytes were analyzed by double immunofluorescence with antibodies against vinculin, a specific focal adhesion component (detected with fluorescein isothiocyanate-coupled secondary antibodies; green) and phalloidin, for actin cable visualization (rhodamine-coupled; red). Samples were analyzed by confocal microscopy, and green and red images were superimposed. Focal adhesions and actin cables were readily detectable in control keratinocytes, whereas they were totally disrupted as early as 30 h after TPA exposure (Fig. 4, upper panels). By contrast, little or no disruption of actin cables and focal adhesions were induced by TPA in cells that had been pretreated with TGF-beta 3 for 18 h (Fig. 4, lower panels). Relative to TGF-beta 3, pretreatment with TGF-beta 1 had more limited counteracting effects on TPA-induced disruption of focal adhesions and disassembly of actin cables (Fig. 4, middle panels). In parallel with these findings, TPA treatment induced rounding up of keratinocytes by 6 h (Fig. 5A) and detachment from the dish by 24 h (Fig. 5B). These effects were almost totally prevented by pretreatment with TGF-beta 3, whereas TGF-beta 1 exerted a more partial protection (Fig. 5).


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Fig. 4.   TPA-induced disruption of focal adhesions and actin cables and counteracting effects of TGF-beta 3. Primary mouse keratinocytes were either left untreated (Ctrl) or treated with TGF-beta 1 or TGF-beta 3 for 18 h before exposure to Me2SO vector alone or TPA (100 ng/ml) for an additional 30 h. Cells were analyzed by double immunofluorescence with antibodies against vinculin and fluorescein isothiocyanate-conjugated secondaries (green) and Texas red-conjugated phalloidin (red). Samples were analyzed by confocal microscopy, and green and red images were superimposed so that sites of overlapping staining are visualized as yellow. Bar, 4 µm. Photographs were exposed for identical amounts of time. Similar results were observed at 2 h of TPA treatment and were confirmed in a second independent experiment.


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Fig. 5.   TPA-induced cell detachment and counteracting effects of TGF-beta 3. Primary mouse keratinocytes were either left untreated (Ctrl) or treated with TGF-beta 1 or TGF-beta 3 for 18 h before exposure to TPA (100 ng/ml) for either 6 h (A) or 24 h (B). Panel A, contrast phase microscopy (10×) illustrating the differential rounding up of cells after 6 h of TPA treatment, depending on whether or not cells were pretreated with TGF-beta 1 or TGF-beta 3. Panel B, keratinocyte cell detachment at 24 h of TPA treatment. Cells that had remained attached to the dish were trypsinized and counted, in parallel with those that had detached in the culture medium. Each condition was tested on duplicate dishes, and values are expressed as ratios of detached versus detached cells recovered from each dish.

5) Specific Inhibitory Effects of TGF-beta 3 Versus TGF-beta 1 on TPA-induced JNK Activation-- Previous work has demonstrated that loss of keratinocyte cell adhesion can trigger JNK kinase activation (26), and preferential activation of the JNK versus ERK kinase can be a critical determinant of apoptosis (27). Differential modulation of these pathways could provide a mechanism for the specific protective effects of the TGF-beta 3 versus TGF-beta 1 isoforms on TPA-induced cell death. To test this possibility, primary keratinocytes were either untreated or treated with TGF-beta 1 or TGF-beta 3 (3 ng/ml) for 18 h followed by exposure to TPA for different lengths of time. Total cell extracts were incubated with beads coupled to the c-Jun protein to immunoprecipitate JNK or with phospho-specific antibody to the ERK1/2 kinase to immunoprecipitate the active form of this kinase. In vitro kinase assays were carried out using recombinant c-Jun or Elk1 proteins as substrates for the JNK and ERK1/2 kinases, respectively. In vitro kinase activity was normalized by the total amounts of JNK and ERK proteins present inside the cells, as determined by immunoblotting of the same cell extracts with antibodies against these proteins. A representative experiment is shown in Fig. 6A, whereas Fig. 6B illustrates the composite quantification of three independent experiments. As reported for other cell types, treatment of mouse primary keratinocytes with TPA resulted in the early induction of JNK kinase activity, which returned to basal level or even below by 4-8 h of treatment. Activity of the related ERK kinase was also induced in the TPA-treated keratinocytes, with a similar time course of activation. Keratinocytes pretreated with TGF-beta 1 showed the same pattern of JNK activation in response to TPA as the control, with the exception that the basal level of JNK kinase activity appeared elevated in these cells even before TPA treatment. ERK kinase was induced in the TGF-beta 1-pretreated keratinocytes after exposure to TPA, but ERK activity levels remained consistently lower than in controls. In contrast, elevation of JNK activity in response to TPA was significantly delayed and/or suppressed in the TGF-beta 3-treated keratinocytes, whereas ERK activity was induced by TPA to a similar if not greater extent than in the controls. Thus, the relative ratio of JNK versus ERK activation was substantially lower in the TGF-beta 3 versus TGF-beta 1-treated keratinocytes (Fig. 6B), consistent with the specific counteracting effects of the first isoform on TPA-induced cell death.


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Fig. 6.   JNK and ERK1/2 activation in TPA-treated keratinocytes and differential modulatory effects of the TGF-beta 3 versus TGF-beta 1 isoforms. Panel A, primary mouse keratinocytes were either left untreated (Control, Ctl) or treated with TGF-beta 1 or TGF-beta 3 for 18 h before exposure to TPA (100 ng/ml) for the indicated amounts of time. Total cell extracts were normalized for protein amounts and assayed for JNK and ERK1/2 in vitro kinase activity using c-Jun and Elk1 proteins as substrates as described under "Experimental Procedures." The same extracts were analyzed for total JNK and ERK protein levels by immunoblotting with the corresponding specific antibodies. Panel B, composite result of three independent experiments performed as described in panel A. Quantitation was achieved by densitometric scanning of the autoradiographs and normalization for total JNK and ERK protein levels. In the first two panels, values are expressed as -fold of induction relative to basal kinase activity in the untreated controls. In the right panel, the relative ratio of JNK versus ERK kinase activity was calculated.


    DISCUSSION

Previous data from a number of laboratories have shown that the TGF-beta 3 isoform is preferentially produced by dermal fibroblasts and not keratinocytes, and that in the skin, the presence of this factor in the epidermis is likely because of diffusion from the underlying dermis (9, 11, 12, 14). The present findings establish a unique function of TGF-beta 3 not shared by TGF-beta 1 as a specific isoform able to protect keratinocytes from the death-inducing effects of an agent such as TPA. These findings help to explain a long-standing discrepancy between the in vivo effects of TPA (induction of epidermal hyperplasia) and the effects of this agent on cultured keratinocytes (induction of growth arrest, differentiation, and as we have shown here, increased cell death). Because keratinocytes produce very little if any TGF-beta 3, these cells are intrinsically sensitive to the death-inducing effects of TPA but could be protected from these effects in vivo by the dermally produced TGF-beta 3.

Apoptosis is typically associated with a multistage process of DNA fragmentation, cell shrinking, and lack of an inflammatory response (21, 28, 29). A necrosis reaction can also be associated with random DNA fragmentation, but it involves cell swelling and a significant inflammatory reaction (29, 30). The histological findings of the TGF-beta 3 knockout epidermis treated with TPA exhibited mixed features of apoptosis and necrosis. An increasing number of variant cell deaths have been described to which the dichotomy apoptosis/necrosis cannot be applied, and even biochemically, the distinction is less clear than expected (31). For instance, it has recently been shown that well established inhibitors of apoptosis such as Bcl-2 and ICE inhibitors can also suppress classical necrosis (32). In addition to cell death in the epidermis, the TPA-treated TGF-beta 3 knockout grafts showed evidence of dermal necrosis as well as microangiopathy. Our in vitro data are consistent with the alterations of the epidermis being a primary consequence of keratinocyte exposure to TPA in the absence of the TGF-beta 3-protective function. However, alterations in the dermis and blood vessels could also contribute to keratinocyte cell death in the overlying epidermis.

Under basal conditions, grafted epidermis of TGF-beta 3 knockout mice was histologically normal, and no increased apoptosis was observed. However, levels of TGF-beta 3 were reduced but not totally absent in these grafts, consistent with this factor diffusing to some extent from the neighboring skin in amounts that might be sufficient for normal skin maturation

The mature TGF-beta 1, -beta 2, and -beta 3 isoforms share a >70% amino acid sequence identity, including conservation of the 8 cysteine residues that contribute to the correct folding of the monomers and the additional cysteine residue involved in subunit dimerization (33). The sequence diversity among individual isoforms has been maintained throughout evolution, suggesting that they may have intrinsically distinct functions (34). In fact, although all three isoforms bind to type I and type II TGF-beta receptors and exert similar effects on many cell types, in certain cases they behave selectively. For instance, TGF-beta 2 exerts little if any growth inhibitory effects on endothelial cells (35), and this may be because of the lack of binding of TGF-beta 2 to endoglin, a major surface TGF-beta -binding protein that coexists with the type I and type II TGF-beta receptors on endothelial cells (36). Similarly, TGF-beta 3, unlike TGF-beta 1 and -beta 2, fails to bind to heparin and to liver heparan sulfate (37). These polysaccharides potentiate the biological activity of TGF-beta 1 by antagonizing the binding of this isoform and its inactivation by alpha 2 macroglobulin, a major TGF-beta -binding protein found in serum and the intercellular space (38). The differential binding of the TGF-beta isoforms to heparin has been related to two specific basic residues at positions 26 and 60 in TGF-beta 1 and -beta 2, which are replaced by neutral ones in TGF-beta 3 (37). In primary mouse keratinocytes grown under our conditions, TGF-beta 1 and TGF-beta 3 were found to exert similar growth inhibitory effects and to induce a TGF-beta -responsive promoter (3TP-luc) to a similar extent.2 This is consistent with the fact that these factors bind type I and type II TGF-beta receptors with similar affinity (1), and are therefore expected to trigger similar downstream reponses. However, TGF-beta 1 treatment was by itself sufficient to increase the fraction of TUNEL positive keratinocytes, whereas no such effect was elicited by TGF-beta 3. More importantly, there was a substantial difference in the way pretreatment with TGF-beta 3 but not TGF-beta 1, protected keratinocytes from TPA-induced cell death. An attractive hypothesis is that TGF-beta 3, besides triggering type I and type II receptors to a similar extent as TGF-beta 1, binds specifically to other proteins on the keratinocyte cell surface, thus affecting behavior of these cells in additional manners.

The protective function of TGF-beta 3 on TPA-induced cell death is not because of general suppression of TPA signaling, as ERK1/2 activation occurred to a similar if not greater extent in TGF-beta 3 treated versus control keratinocytes. Instead, TGF-beta 3 treatment led to a significant reduction in TPA-induced JNK activity, whereas increased rather than decreased JNK activity was observed after treatment of the same cells with TGF-beta 1. Activation of ERK has been connected with growth stimulation of cells in response to a variety of stimuli, whereas induction of JNK has been closely related to apoptosis (39). In epithelial cells, one important inducer of JNK activity (and apoptosis) is loss of cell attachment to the substrate (40). The rapid disruption of stress fibers and focal adhesions caused by TPA treatment was found to be effectively counteracted by TGF-beta 3, thus providing an attractive explanation for the protective effects of this isoform against TPA-induced cell death. A specific role of TGF-beta 3 in promoting epithelial cell adhesion is consistent with the major phenotype of TGF-beta 3 knockout mice in which cleft palate has been related to reduced adhesion of the epithelial medial edge (6). Future studies will have to further investigate the consequences of this specific function of the TGF-beta 3 isoform in epidermal homeostasis.

    ACKNOWLEDGEMENTS

We thank to Dr. Cythia Magro for reviewing the histological slides and Drs. Caterina Missero, Cathrin Brisken, and Jerry Gross for critical reading of the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants AR39190, CA16038, and CA73796 (to G. P. D.) and in part by the Cutaneous Biology Research Center through the Massachusetts General Hospital/Shiseido Co. Ltd. Agreement.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Present address: Dept. of Dermatology, Stanford University Medical School, Stanford, CA 94305.

parallel To whom correspondence should be addressed: CBRC, MGH-East, 13th St., Charlestown MA 02129. Tel.: 617-724-9538; Fax: 617-724-9572; E-mail: Paolo.Dotto{at}cbrc2.mgh.harvard.edu.

The abbreviations used are: TPA, 12-O-tetradecanoylphorbol-13-acetate; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP biotin nick end-labeling; JNK, c-Jun N-terminal kinase.

2 J. Li, K. Foitzik, E. Calautti, H. Baden, T. Doetschman, and G. Paolo Dotto, unpublished observations.

    REFERENCES
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Abstract
Introduction
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