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DIFFERENTIAL MOTOGENIC AND BIOSYNTHETIC RESPONSE OF FETAL AND ADULT SKIN FIBROBLASTS TO TGF-β ISOFORMS
DIFFERENTIAL MOTOGENIC AND BIOSYNTHETIC RESPONSE OF FETAL AND ADULT SKIN FIBROBLASTS TO TGF-b ISOFORMS Ian R. Ellis and Seth L. Schor Data are presented in this communication comparing fetal and adult fibroblasts with respect to the effects of transforming growth factor b (TGF-b) isoforms (-b1, -b2 and -b3) on cell migration and hyaluronan (HA) synthesis. Cell migration was assessed on three-dimensional native type I collagen substrata. Fetal and adult cells differed in terms of their motogenic response to the three TGF-b isoforms in a manner which was modulated by cell density, i.e.: (1) the migration of subconfluent fetal cells was unaffected by TGF-b1 and -b2, but inhibited by TGF-b3, whilst the migration of subconfluent adult cells was inhibited by all three isoforms, and (2) the migration of confluent fetal cells was inhibited by all three TGF-b isoforms, whilst the migration of confluent adult cells was unaffected by TGF-b1 and -b2, but stimulated by TGF-b3. This diverse pattern of motogenic response to the three TGF-b isoforms was paralleled by similar effects on HA synthesis (i.e. inhibition, no effect or stimulation). Linear regression analysis revealed a significant correlation between cell migration and total HA synthesis (r2 = 0.861; P Q 0.0001). Gel filtration chromatography of cell-produced HA indicated that the effects of TGF-b isoforms on total HA synthesis reflected alterations in the relative production of high molecular mass species (Mr q 106). Taken together with previously published data, these observations indicate that (1) fetal and adult fibroblasts exhibit distinct responses to the three TGF-b isoforms with respect to both cell migration and HA synthesis, (2) cellular response to the TGF-b isoforms is modulated by cell density, and (3) TGF-b3 is the only isoform which stimulated cell migration and HA synthesis (with confluent adult cells). 7 1998 Academic Press Limited
Fetal and adult fibroblasts differ with respect to a number of phenotypic characteristics, including the synthesis of specific isoforms of matrix macromolecules,1 the production of cytokines2,3 and cellular response to these two classes of effector molecules.4–6 In this regard, we have recently reported that: (1) the migration of confluent fetal and adult fibroblasts is differentially affected by epidermal growth factor (EGF), transforming growth factor b1 (TGF-b1), and platelet-derived growth factor (PDGF), and (2) the synthesis of hyaluronan (HA) by fetal and adult fibroblasts is also differentially affected by these cytokines.7 The TGF-b family of cytokines are multifunctional regulators of various aspects of cell behaviour, including proliferation,8 matrix synthesis9 and cell migration.10–13 There are three principal mammalian
From the Department of Dental Surgery and Periodontology, The Dental School, University of Dundee, Dundee DD1 4HR, UK Correspondence to Seth Schor, E-mail: SLSchor.ITS.dundee.ac.uk Received 12 June 1997; accepted for publication 22 September 1997 7 1998 Academic Press Limited 1043–4666/98/040281 + 09 $25.00/0/ck970294 KEY WORDS: TGF-b/ fibroblasts/ migration/ hyaluronan/ wound healing CYTOKINE, Vol. 10, No. 4 (April), 1998: pp 281–289
isoforms of TGF-b.14 These commonly exert similar effects on cell behaviour,15 although several studies have revealed isoform-specific differences in both target cell responsiveness and mode of action.16 These observations are consistent with the differential expression of TGF-b isoforms during development17,18 and the resultant speculation that they may fulfill distinct functions in embryogenesis, as well as in the adult organism.19 Our previous work has indicated that the effect of TGF-b1 on fibroblast migration is dependent upon both developmental stage (fetal vs adult) and cell density.7,12,20 The objective of the present study has consequently been to extend these observations to include other members of the TGF-b family of cytokines.
RESULTS Fetal and adult fibroblasts were plated at subconfluent and confluent cell densities onto the surface of 3D collagen gel substrata in the absence (control) and presence of different concentrations of TGF-b1, TGF-b2, and TGF-b3. Data obtained with one representative fetal and one adult fibroblast line confirm our previous observations regarding the 281
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30 A
C
B
D
Percentage cells in gel matrix
20 10 0 15 10 5 0
0.1
1.0 10.0 0.1 1.0 Concentration TGF-β (ng/ml)
10.0
Figure 1. The effects of TGF-b isoforms on the migration of fetal (A,B) and adult (C,D) skin fibroblasts. Data are presented concerning the effects of TGF-b1 (q), TGF-b2 (<) and TGF-b3 (Q) on the migration of one fetal and one adult human skin fibroblast line. Cells were plated onto collagen gels at subconfluent (A,C) and confluent (B,D) cell densities and the effects of the different TGF-b isoforms on their subsequent migration into the three- dimensional gel matrix measured after a 4-day incubation period (as indicated in Materials and Methods). Data are expressed as the percentage of total cells present within the gel matrix (mean 2 SD of five replicate experiments). Dashed line indicates the percentage of cells present in the gel matrix of control cultures.
differential and density-dependent effects of TGF-b1 on these two cell populations (Fig. 1), i.e. TGF-b1 had no significant effect on the migration of subconfluent fetal fibroblasts, inhibited the migration of confluent fetal fibroblasts and subconfluent adult cells, and had no effect on confluent adult cells. Our data further TABLE 1.
Fetal
Adult
indicate that TGF-b2 exerts essentially the same effects as TGF-b1 on fibroblast migration, although there are quantitative differences with respect to the concentration of these isoforms required to achieve maximal inhibition of confluent fetal fibroblast migration. In contrast, TGF-b3 differed from the other two isoforms in that it inhibited the migration of subconfluent fetal fibroblasts and stimulated the migration of confluent adult fibroblasts. TGF-b3 did not differ from the -b1 and -b2 isoforms regarding its inhibitory effect on the migration of subconfluent adult and confluent fetal fibroblasts. Data summarizing the effects of TGF-b1, -b2 and -b3 on the migration of three fetal and three adult fibroblast lines are presented in Table 1 (expressed in terms of absolute migration) and Figure 2 (expressed in terms of migration relative to control). TGF-b isoforms were used at optimal concentrations, i.e. 10 ng/ml TGF-b1 and TGF-b2, and either 0.1 or 10 ng/ml TGF-b3. These data confirm that the motogenic activity of TGF-b1 and -b2 differ from that of TGF-b3 and are dependent upon both developmental stage of the target cells and their degree of confluence. The effects of TGF-b isoforms on HA synthesis by the same three fetal and adult fibroblast lines are summarized in Table 2 (absolute levels of [3 H]glucosamine incorporation) and Figure 3 (incorporation relative to control). These data parallel those relating to cell migration. Linear regression analysis (Fig. 4) of data obtained under each combination of cell type, cell density and TGF-b isoform confirms that there is a good correlation between cell migration and total HA synthesis under all experimental conditions
Effect of TGF-b isoforms on the migration of adult and fetal fibroblasts Control
TGF-b1
TGF-b2
TGF-b3
Subconfluent
F104 F105 FS6 Mean 2 SD
19.1 2 1.6 15.6 2 0.9 18.2 2 0.6 17.6 2 1.5
16.9 2 0.9 17.0 2 1.1 15.9 2 0.4 16.4 2 0.4
18.1 2 0.4 17.0 2 0.1 16.3 2 0.0 17.1 2 0.7
5.0 2 0.2 6.3 2 0.2 6.0 2 1.1 5.8 2 0.6
Confluent
F104 F105 FS6 Mean 2 SD
11.2 2 0.8 14.3 2 1.1 16.6 2 0.3 14.0 2 2.2
4.7 2 0.7 6.5 2 1.1 2.9 2 0.4 4.7 2 1.5
2.2 2 0.5 5.9 2 1.0 3.4 2 0.2 3.8 2 1.5
2.0 2 0.7 3.9 2 1.0 3.0 2 0.6 3.0 2 0.8
Subconfluent
FSF37 FSF44 SK319 Mean 2 SD
15.6 2 0.3 25.4 2 0.8 20.8 2 1.0 20.6 2 4.0
6.5 2 0.1 5.6 2 0.1 8.2 2 1.1 6.8 2 1.1
5.9 2 0.6 13.7 2 2.4 9.1 2 0.3 9.6 2 3.2
6.8 2 0.3 8.7 2 0.2 7.6 2 0.9 7.7 2 0.8
Confluent
FSF37 FSF44 SK319 Mean 2 SD
2.0 2 0.1 1.9 2 0.1 2.3 2 0.5 2.1 2 0.2
1.9 2 0.3 1.6 2 0.4 2.1 2 0.9 1.9 2 0.2
2.0 2 0.1 1.6 2 0.4 3.3 2 1.0 2.3 2 0.7
8.4 2 1.0 7.1 2 0.2 9.6 2 0.5 8.4 2 1.0
Data are presented concerning the effects of optimal concentrations of TGF-b1 (10 ng/ml), TGF-b2 (10 ng/ml) and TGF-b3 (0.1 or 10 ng/ml) on the migration of several adult and fetal fibroblast lines. Cell migration was determined microscopically on day 4, as described in Materials and Methods, and expressed as the percentage of cells within the gel matrix.
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1.5
1.5
Relative migration
A
Gel filtration chromatography was used to analyse the effects of TGF-b isoforms on the polydispersity of HA synthesized by representative fetal and adult fibroblast lines (Figs 5 and 6, respectively). These data indicate that the cell-density dependent effects of TGF-b isoforms on total HA synthesis (inhibitory, no effect, or stimulatory) result from a corresponding effect on the relative production of high molecular species of HA.
C
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0.5
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0
1.5 B
4
D
DISCUSSION
3
1.0
2 0.5 1 0
β1
β2
0 β3 β1 TGF-β isoforms
β2
β3
Figure 2. The effects of TGF-b isoforms on the migration of fetal (A,B) and adult (C,D) skin fibroblasts. Summary of data obtained with three lines of fetal and three lines of adult skin fibroblasts. Cells were plated at subconfluent (A,C) and confluent (B,D) densities and incubated with concentrations of the TGF-b isoforms which elicited maximal response (0.1 or 10 ng/ml TGF-b3 and 10 ng/ml TGF-b1 and TGF-b2), as indicated in Figure 1. Data are expressed as the percentage of cells within the gel matrix relative to control. Dashed line indicates control level of migration.
(r2 = 0.861; P Q 0.0001). The diverse effects of TGF-b isoforms on fetal and adult fibroblasts in terms of both cell migration and HA synthesis are summarized in Table 3. TABLE 2.
Fetal
Adult
The objective of this study has been to compare the response of fetal and adult skin fibroblasts to TGF-b isoforms with respect to cell migration and HA synthesis. Our results indicate that fetal and adult fibroblasts exhibit distinct responses to these isoforms, and that these differences are dependent upon: (1) isoform type; and (2) cell density.
Dependence on developmental state Fetal and adult fibroblasts cultured at identical cell densities responded differently to TGF-b isoforms in terms of both cell migration and HA synthesis. Our data indicate that: (1) the migration of subconfluent fetal cells was unaffected by TGF-b1 and -b2, but inhibited by TGF-b3, whilst the migration of subconfluent adult cells was inhibited by all three isoforms; and (2) the migration of confluent fetal cells was inhibited by all three TGF-b isoforms, whilst the migration of confluent adult cells was unaffected by TGF-b1 and -b2, but stimulated by TGF-b3. In the absence of exogenous effector molecules, control fetal
Effect of TGF-b isoforms on HA synthesis by adult and fetal fibroblasts Control
TGF-b1
TGF-b2
TGF-b3
Subconfluent
F104 F105 FS6 Mean 2 SD
123.2 2 3.5 136.7 2 2.9 103.9 2 2.1 121.3 2 13.4
118.1 2 10.0 140.6 2 9.6 100.6 2 3.1 119.8 2 16.4
126.5 2 12.6 125.6 2 5.2 101.5 2 9.3 117.9 2 11.6
78.6 2 4.2 95.6 2 1.9 80.6 2 2.5 84.9 2 7.6
Confluent
F104 F105 FS6 Mean 2 SD
120.1 2 12.9 141.7 2 6.3 113.9 2 6.6 125.2 2 11.9
58.9 2 14.3 80.6 2 6.9 59.9 2 26.7 66.5 2 10.0
14.5 2 8.5 60.1 2 5.6 64.7 2 8.4 46.3 2 22.7
53.7 2 23.2 58.5 2 7.5 62.1 2 9.6 58.1 2 3.4
Subconfluent
FSF37 FSF44 SK319 Mean 2 SD
114.0 2 1.9 104.3 2 2.8 100.1 2 2.6 106.2 2 5.9
15.7 2 0.6 11.5 2 2.9 16.0 2 1.9 14.4 2 2.0
18.7 2 3.6 6.6 2 4.5 19.5 2 3.6 14.9 2 5.9
17.0 2 0.5 25.8 2 1.5 11.3 2 0.9 18.0 2 6.0
Confluent
FSF37 FSF44 SK319 35.4 2 4.5
34.2 2 0.3 41.4 2 1.8 30.7 2 2.5 33.5 2 5.1
28.1 2 0.1 40.3 2 2.5 32.1 2 1.6 35.2 2 4.6
34.6 2 0.2 41.1 2 0.2 29.8 2 3.5 85.2 2 9.2
89.2 2 0.2 94.0 2 4.2 72.5 2 1.6
Mean 2 SD
Data are presented concerning the effects of optimal concentrations of TGF-b1 (10 ng/ml), TGF-b2 (10 ng/ml) and TGF-b3 (0.1 or 10 ng/ml) on HA by several adult and fetal fibroblast lines. HA synthesis was determined microscopically on day 4, as described in Materials and Methods, and expressed as dpm [3H]glucosamine incorporated in HA 10 − 5 cells × 103.
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1.5
1.5
4
C
1.0
1.0
0.5
0.5
0
0
1.5
3
Relative HA synthesis
Relative HA synthesis
A
3
2
1
D
B 1.0
2
0
0.5
1
Figure 4.
0
0
The correlation between HA synthesis and cell migration of three fetal and adult lines in the absence and presence of TGF-b1, TGF-b2, and TGF-b3 were analysed by linear regression analysis (r 2 = 0.861; P Q 0.0001).
β1
β2
β1 β3 TGF-β isoforms
β2
β3
Figure 3. The effects of TGF-b isoforms on HA synthesis by fetal (A,B) and adult (C,D) skin fibroblasts. Summary of data obtained with three lines of fetal and three lines of adult skin fibroblasts. Cells were plated at subconfluent (A,C) and confluent (B,D) densities and incubated with concentrations of the TGF-b isoforms which elicited maximal response (0.1 or 10 ng/ml TGF-b3 and 10 ng/ml TGF-b1 and TGF-b2), as indicated in Figure 1. Data are expressed as [3H]glucosamine incorporated into HA relative to control. Dashed line indicates control level of HA synthesis.
and adult fibroblasts also differ with respect to their migratory behaviour and HA synthesis.21,22 These intrinsic differences appear to result from their differential production of motogenic cytokines2,3,24–27 and the modulation of HA synthesis by these effector molecules.7,12,28,29 Fetal and adult fibroblasts also differ with respect to a number of other biochemical characteristics of potential relevance to migration, including the activity of matrix synthesizing enzymes,30 the synthesis of specific isoforms of matrix glycoproteins1,31 and the secretion of matrix degrading enzymes.32,33
Dependence on TGF-b isoform TGF-b isoforms have commonly been found to elicit similar effects upon cell behaviour in vivo34 and in vitro,15,35–37 although there may be significant differences in their relative potencies.16 With specific reference to cell migration, Parekh et al.38 observed that TGF-b1, -b2 and -b3 all stimulated neutrophil migration in the transmembrane assay. Data presented in this communication indicate that the different TGF-b isoforms elicited distinct effects upon fibroblast migration and HA synthesis. Interestingly, TGF-b1 and -b2 were always similar in
1
2 3 Relative migration
4
5
Correlation between cell migration and HA synthesis.
their biological activity (either inhibitory or inactive); the bioactivity of TGF-b3 differed from that of the other two isoforms in subconfluent fetal fibroblasts (where it inhibited cell migration and HA synthesis) and in confluent adult cells (where it stimulated both cellular activities). These observations are consistent with previous reports documenting qualitative differences in the biological activity of TGF-b isoforms.39,40 In this regard, it is of interest that there are both temporal and spatial differences in the expression of the three TGF-b isoforms at both the protein and mRNA levels during embryonic development.17,18,41 Such differences suggest that TGF-b isoforms fulfil different functions and may continue to do so in the adult.19
Dependence on cell density The effects of all three TGF-b isoforms were modulated by cell density. Indeed, with the exception of the effect of TGF-b3 on fetal fibroblasts, all TGF-b isoforms elicited distinct effects at subconfluent and
TABLE 3. Effects of TGF-b isoforms on fetal and adult fibroblasts Fetal
Adult
Subconfluent
TGF-b1 TGF-b2 TGF-b3
t t 4
4 4 4
Confluent
TGF-b1 TGF-b2 TGF-b3
4 4 4
t t 3
Data summarizing the effects of TGF-b isoforms on both cell migration and HA synthesis. Key: 4, inhibition; t, no effect; 3, stimulation.
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75 A
Vdb
Vt
E
Vdb
Vt
50
25
0
75 B
F
C
G
D
H
[3H]HA (dpm/105 cells, × 10–3)
50
25
0
75
50
25
0
75
50
25
0
10
20
30
40
50 0 10 Fraction number
20
30
40
50
Figure 5. Effect of TGF-b isoforms on the size class distribution of HA synthesized by subconfluent (A,B,C,D) and confluent (E,F,G,H) fetal fibroblasts. Cells were incubated with concentrations of TGF-b isoforms which elicited maximal response, as indicated in Fig. 1. The polydispersity of HA synthesized by fetal fibroblasts plated on the surface of a 3D collagen gel was analysed using Sepharose CL-2B column chromatography as described in the Materials and Methods. Vdb indicates the point at which dextran blue was eluted from the column and is an estimate of the void volume; Vt indicates the total column volume.
confluent cell densities (summarized in Table 3). The inhibition of fibroblast proliferation by TGF-b1 has previously been reported to be modulated by cell density.42 Cell density also affects the behaviour of control fetal and adult fibroblasts with respect to their migratory activity21 and HA synthesis.22 The densitydependent effects of the different TGF-b isoforms on
fetal and adult cells reported here presumably reflect the differential response of cells in different physiological states with respect to their intrinsic level of cell migration and HA synthesis, as well as other potentially relevant parameters including the synthesis of cytokines and matrix molecules, their respective cell surface receptors and matrix degrading enzymes.
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A
Vdb
15
Vt
30
E
Vdb
Vt
10
20 5
10 0
[3H]HA (dpm/105 cells, × 10–3)
40
10
20
30
40
50
0
10
20
30
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20
30
40
50
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20
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50 0 10 Fraction number
20
30
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50
15
B
30
F
10
20 5
10 0 40
10
20
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40
50
0 15
C
30
G
10
20 5
10 0 40
10
20
30
40
50
0 15
D
30
H
10
20 5
10 0
10
20
30
40
Figure 6. Effect of TGF-b isoforms on the size class distribution of HA synthesized by subconfluent (A,B,C,D) and confluent (E,F,G,H) adult fibroblasts. Cells were incubated with concentrations of TGF-b isoforms which elicited maximal response, as indicated in Fig. 1. The polydispersity of HA synthesized by adult fibroblasts plated on the surface of a 3D collagen gel was analysed using Sepharose CL-2B column chromatography as described in the Materials and Methods. Vdb indicates the point at which dextran blue was eluted from the column and is an estimate of the void volume; Vt indicates the total column volume. (A,E), control; (B,F), TGF-b1; (C,G), TGF-b2; (D,H), TGF-b3.
Correlation between HA synthesis and cell migration: implications for cell behaviour during development and in the adult Data presented in Figure 4 indicate a close correlation between cytokine-induced alterations in cell migration and HA synthesis. A similar correlation has previously been observed with a number of other motogenic cytokines.7 In this regard it is of particular interest that there is a significant spatial and temporal correlation between TGF-b expression and HA
concentration during embryonic development;43 these observations led the authors to speculate that TGF-b modulates HA synthesis and that this alteration in matrix may in turn mediate many of the biological effects of TGF-b. This postulated mechanistic link is consistent with the reported effect of HA on the migration of a number of cell types.44 These effects of HA on cell migration are mediated by interaction with its receptors, CD-44 and RHAMM.45,46 CD-44 is a complex family of related molecules produced by alternative splicing.47 Individual CD-44 isoforms
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display developmentally regulated differences in both cell expression and ligand affinity. Signal transduction elicited by ligation of RHAMM which may affect cell migration include tyrosine phosphorylation of the focal adhesion kinase pp125FAK.48,49 Related studies have indicated that the effects of several cytokines on cell migration may be both mediated and modulated by the extracellular matrix.38,50 All three TGF-b isoforms appeared to affect the synthesis of high molecular mass HA. A similar preferential modulation of high molecular mass HA has been reported for a number of cytokines, including TGF-b1.7 Our previous data suggest that alterations in this particular size class of HA are primarily responsible for HA-mediated changes in cell motility.7,12,20 The differential effects of TGF-b isoforms -b1, -b2 and -b3 on the migration of fetal and adult fibroblasts reported here may be relevant to differences in fetal and adult wound healing and the manner in which this is affected by members of the TGF-b family. Adult wound healing is a ‘‘reparative’’ process, commonly accompanied by scar formation;51 in contrast, dermal wound healing in the early gestation fetus is regenerative in nature and essentially scar-free.52,53 TGF-b1 and -b2 have been detected in adult wounds, whilst TGF-b3 is the principal isoform found in fetal wounds.54 Treatment of adult wounds with either neutralising antibodies for TGF-b1 and -b2, or exogenous application of TGF-b3, significantly reduced scar formation in healing adult wounds;55 conversely, application of TGF-b1 to fetal wounds induced excessive scar formation.56
MATERIALS AND METHODS Reagents Human platelet-derived TGF-b1, TGF-b2, and human recombinant TGF-b3 was either a generous gift from Dr A. Roberts (NIH, MD) or purchased from R&D Systems, Ltd. (Cowley, Oxford, UK). -[3 H]glucosamine hydrochloride (22 Ci/mmol) was obtained from Amersham International plc (Amersham, Bucks., UK). Hyaluronan (type 1, sodium salt), chondroitin-6-sulfate and hyaluronate lyase were obtained from Sigma Chemical Co. (Poole, Dorset, UK). Ultragold scintillation fluid was obtained from Canberra Packard (Pangbourne, Berkshire, UK). Eagle’s Minimal Essential Medium (MEM), donor calf serum, sodium pyruvate, glutamine, non-essential amino acids, antibiotics and tissue culture plastic dishes were obtained from Gibco BioCult (Paisley, UK). All other chemicals were obtained from BDH Chemicals (Poole, Dorset, UK) or Sigma Chemical Co. (Poole, Dorset, UK).
Cell culture and preparation of collagen gels The fetal and adult fibroblast lines used in this study were established in our laboratory by explant culture from
skin biopsies.57 Stock cultures were maintained in Eagle’s MEM supplemented with 15% (V/V) donor calf serum, 1 mM sodium pyruvate, 2 mM glutamine, non-essential amino acids at 37°C in a moist atmosphere containing 5% CO2. Cells were grown on 90 mm plastic tissue culture Petri dishes and passaged at a split ratio of 1:5 upon reaching confluence 7–10 days after plating. All experiments were performed with fibroblasts between passage 10–18. Cultures used in this study were shown to be free of mycoplasma contamination by staining with Hoechst 33256. Type I collagen was extracted from rat tail tendons in 3% acetic acid, dialysed for 2 days against distilled water, diluted to 2 mg/ml and used to make 2 ml collagen gels in 35 mm plastic tissue culture dishes as previously described.58
Migration assay Collagen gels were overlaid with 1 ml of either SF-MEM (controls) or serum-free MEM containing 4× the final concentration of cytokine. Confluent stock cultures were trypsinized, pelleted by centrifugation, resuspended in growth medium containing 20% donor calf serum at a concentration of either 2.5 × 104 cells/ml (subconfluent) or 2.5 × 105 cells/ml (confluent) and 1-ml aliquots of this inoculum were pipetted onto the cultures. Considering the 2-ml volume of the collagen gel, the 1-ml medium overlay and the 1-ml cell inoculum, this procedure gives a final concentration of 5% serum in both control and cytokinecontaining cultures. The cultures were incubated for 4 days and the percentage of fibroblasts found within the 3D gel matrix was then ascertained by microscopic observation of 15–20 randomly selected fields, as previously described.50 Replicate gels were counted for each experimental condition.
Metabolic labelling and determination of total labelled HA Glycosaminoglycans (GAGs) synthesized by the fibroblasts were metabolically labelled by incubating cultures with 2.5 mCi/ml of [3 H]glucosamine. These cultures were separated into medium and pericellular fractions, as previously described by Ellis et al.12 In this protocol, the medium and gel were transferred to a centrifuge tube and spun at 1000 × g for 20 min at 4°C. The medium was removed and the compacted gel was resuspended in 2 ml of phosphatebuffered saline. Centrifugation was repeated, and the supernatant was combined with the previous one to form the medium fraction. The pericellular fraction was obtained by extracting the cell layer with 2 ml 50 mM Tris–HCl, 4 M guanidinium chloride, pH 7.4, for 24 h at 4°C. The insoluble material was removed by centrifugation at 12 000 × g for 2 min in a microcentrifuge and the supernatant was collected as the pericellular fraction. The medium and pericellular fractions were dialysed against 0.1 M sodium acetate, 5 mM EDTA, pH 5.5 for 48 h (with two changes per day). The dialysate volumes were measured and six 200-ml aliquots were taken of each sample. 20 ml of Streptomyces sp. hyaluronidase (HA lyase: EC 4.2.2.1), at a final concentration 0.5 U/ml in acetate buffer, was added to three of these, whilst 20 ml of acetate buffer was added to the remaining three
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controls. All samples were incubated for 17 h at 37°C, then boiled for 1 min to inactivate the enzyme and unlabelled carrier GAG added (30 ml 1% chondroitin sulfate/0.5% hyaluronate in PBS). The undigested GAGs were precipitated with 4 volumes of 1.3% potassium acetate in 100% ethanol at −20°C for 3 h.59 The precipitate was sedimented by centrifugation at 12 000 × g for 5 min, the supernatant removed and the pellet washed in 1 ml 75% ethanol and sedimented as above. The supernatant was again removed and the pellet redissolved in 100 ml of 1 M sodium hydroxide and boiled for 3 min. The sodium hydroxide was neutralized with 100 ml of 1 M hydrochloric acid and radioactivity determined using a Packard scintillation counter. The activity (dpm) associated with HA was taken as the difference between the counts in the control and enzyme digested preparations. We consistently found that less than 5% of the total synthesized HA was present in the pericellular fraction under all experimental conditions; data are consequently only presented for HA present in the medium fraction. We have previously confirmed the reliability of estimating HA synthesis by radiolabel incorporation by demonstrating the close correlation between the results obtained by this method and biochemical determination of chemical mass.22
Molecular size distribution of labelled HA The molecular size distribution of labelled material was analysed by Sepharose CL-2B gel filtration chromatography of 200-ml aliquots; columns had an internal diameter of 10 mm, contained 25 ml of gel and were equilibrated with 50 mM Tris–HCl, 4 M guanidinium chloride, pH 7.4. Samples of labelled medium and pericellular fractions were first exhaustively dialysed against 50 mM sodium acetate, 10 mM NaCl, pH 5.5. Representative aliquots were digested prior to chromatography with 2 U of Streptomyces sp. hyaluronidase for 17 h in order to identify the radiolabelled HA. The column was eluted with Tris/guanidinium hydrochloride buffer at 6 ml/h. Fractions (0.5 ml) were collected and radioactivity determined with a Packard scintillation counter. VDB and Vt were determined by the elution of dextran blue and sodium dichromate, respectively. The relative proportions of high (empirically defined as q2 × 106 kDa) and low molecular weight HA were estimated by cutting out and weighing the peaks in the radioactivity profile associated with the void (estimated by VDB) and included volumes, respectively.
Statistical analysis All statistical tests were performed using Prism 2 graphics software (GraphPad Software, San Diego, California). The two-tailed t-test was employed.
Acknowledgements This work has been supported by a grant from the Wellcome Trust.
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REFERENCES 1. Matsura H, Hakomori SI (1985) The oncofetal domain of fibronectin defined by monoclonal antibody FDC-6 its presence in fibronectin from fetal and tumor tissues and its absence from normal adult tissues and plasma. Proc Natl Acad Sci USA 82:6517–6521. 2. Grey AM, Schor AM, Rushton G, Ellis I, Schor SL (1989) Purification of the migration stimulating factor produced by fetal and breast cancer patient fibroblasts. Proc Natl Acad Sci USA 86:2438–2442. 3. Stoker M, Gherardi E, Perryman M, Gray J (1987) Scatter factor is a fibroblast-derived modulator of epithelial cell mobility. Nature 327:239–242. 4. Nathan C, Sporn M (1991) Cytokines in context. J Cell Biol 113:981–986. 5. Schor SL (1994) Cytokine control of cell motility: modulation and mediation by the extracellular matrix. Prog Growth Factor Res 5:223–248. 6. Schor SL, Ellis I, Banyard J, Dolman C, Seneviratne K, Gilbert AD, Chisholm DM (1996) Fetal-like phenotypic characteristics of gingival fibroblasts: Potential relevance to wound healing. Oral Dis 2:155–166. 7. Ellis I, Banyard J, Schor SL (1997) Differential response of fetal and adult fibroblasts to cytokines: cell migration and hyaluronan synthesis. Development 124:1593–1600. 8. Roberts AB, Anzano MA, Wakefield LM, Roche NS, Stern DF, Sporn MB (1985) Typeb transforming growth factor: A bifunctional regulator of cellular growth. Proc Natl Acad Sci USA 82:119–123. 9. Roberts AB, Heine UI, Flanders KC, Sporn MB (1990a) Transforming growth factor-b: Major role in regulation of extracellular matrix. Ann New York Acad Sci 580:225–232. 10. Wahl SM, Hunt DA, Wakefield LM, McCartney-Francis N, Wahl LM, Roberts AB, Sporn MB (1987) Transforming growth factor b induces monocyte chemotaxis and growth factor production. Proc Natl Acad Sci USA 84:5788–5792. 11. Postlethwaite AE, Keski-Oja J, Moses HL, Kang AH (1987) Stimulation of chemotactic migration of human fibroblasts by TGF-b. J Exp Med 165:251–256. 12. Ellis I, Grey AM, Schor AM, Schor SL (1992) Antagonistic effects of TGFb-1 and MSF on fibroblast migration and hyaluronic acid synthesis: Possible implications for dermal wound healing. J Cell Sci 102:447–456. 13. Adelmann-Grill BC, Wach F, Cully Z, Hein R, Krieg T (1990) Chemotactic migration of normal dermal fibroblasts towards EGF and its modulation by PDGF and TGF-b. Eur J Cell Biol 51:322–326. 14. Massague J (1990) The transforming growth factor b family. Annu Rev Cell Biol 6:597–641. 15. Graycar JL, Miller DA, Arrick BA, Lyons RM, Moses HL,Derynck R (1989) Human transforming growth factor-b3: Recombinant expression purification and biological activities in comparison with transforming growth factors-b1 and -b2. Mol Endocrinol 3:1977–1986. 16. Roberts AB, Kondaiah P, Rosa F, Watanabe S, Good P, Danielpour D, Roche NS, Rebbert ML, Dawid IB, Sporn MB (1990) Medoderm induction in Xenopus laevis distinguishes between the various TGFb isoforms. Growth Factors 3:277–286. 17. Pelton RW, Dickson ME, Moses HL, Hogan BLM (1990) In situ hybridization of TGF-b3 RNA expression during mouse development: comparative studies with TGF-b1 and -b2. Development 110:609–620. 18. Akhurst RJ, FitzPatrick DR, Gatherer D, Lehnert SA, Millan FA (1990) Transforming growth factor betas in mammalian embryogenesis. Prog Growth Factor Res 2:153–168. 19. Roberts AB, Sporn MB (1992) Differential expression of TGF-beta isoforms in embryogenesis suggests specific roles in developing and adult tissues. Mol Reprod Dev 32:91–98.
(p.b.e.) — CYTO — 10/4 — PRESS SET — ( 847375 ) —MS 0199 Distinct effect of TGF-b isoforms / 289 20. Ellis IR, Schor SL (1996) Differential effects of TGF-b1 on hyaluronan synthesis by fetal and adult skin fibroblasts: Implications for cell migration and wound healing. Exp Cell Res 228:326–333. 21. Schor SL, Schor AM, Rushton G, Smith L (1985) Adult fetal and transformed fibroblasts display different migratory phenotypes on collagen gels: evidence for an isoformic transition during fetal development. J Cell Sci 73:221–234. 22. Chen J, Grant ME, Schor AM, Schor SL (1989) Differences between adult and foetal fibroblasts in the regulation of hyaluronate synthesis: correlation with migratory activity. J Cell Sci 94:577–589. 23. Clemmons DR (1983) Age dependent production of a competence factor by human fibroblasts. J Cell Physiol 114: 61–67. 24. Schor SL, Schor AM, Grey AM, Rushton G (1988) Fetal and cancer patient fibroblasts produce an autocrine migration stimulating factor not made by normal adult cells. J Cell Sci 90:391–399. 25. Kondo H, Matsuda R, Yonezawa Y (1993) Autonomous migration of human fetal skin fibroblasts into a denuded area in a cell monolayer is mediated by basic fibroblast growth factor and collagen. In Vitro Cellul Dev-Animal 29A:929–935. 26. Kondo H, Yonezawa Y (1995) Fetal-adult transition in terms of their serum dependency and growth factor requirements of human skin fibroblast migration. Exp Cell Res 220:501–504. 27. Locci P, Lilli C, Marinucci L, Baroni T, Pezzetti F, Becchetti E (1993) Embryonic skin fibroblasts release TGF-alpha and TGF-beta able to influence synthesis and secretion of GAG. Cell Mol Biol 39:415–426. 28. Schor SL, Schor AM, Grey AM, Chen J, Rushton G, Grant ME, Ellis I (1989) Mechanism of action of the migration stimulating factor (MSF) produced by fetal and cancer patient fibroblasts: effect on hyaluronic acid synthesis. In Vitro 25:737–746. 29. Heldin P, Laurent TC, Heldin C-H (1989) Effect of growth factors on hyaluronan synthesis in cultured human fibroblasts. Biochem J 258:919–922. 30. Duncan BW, Qian J, Liu X, Bhatnagar R (1992) Regulation of prolyl hydroxylase activity in fetal and adult fibroblasts In: Adzick NS, Longaker MT eds, Fetal wound healing, New York: Elsevier Scientific Press, pp. 303–323. 31. ffrench-Constant C, Van der Water L, Dvorak HF, Hynes RO (1989) Reappearance of embryonic pattern of fibronectin splicing during wound healing in the adult rat. J Cell Biol 109:903–914. 32. Sottile J, Mann DM, Diemar V, Mills AJT (1989) Regulation of collagenase and collagenase mRNA production by early and late passage human diploid fibroblasts. J Cell Physiol 138:281–290. 33. Basset AH, Bellocq JP, Wolf C, Stoll I, Hutin P, Limacher JM, Podhajcer OL, Chenard MP, Rio MC, Chambon P (1990) A novel metalloproteinase gene specifically expressed in stromal cells of breast carcinomas. Nature 348:699–704. 34. Daniel CW, Robinson SD (1992) Regulation of mammary growth and function by TGF-beta. Mol Reprod Dev 32:145–151. 35. Merwin JR, Roberts A, Kondaiah P, Tucker A, Madri J (1991) Vascular cell responses to TGF-beta 3 mimic TGF-beta 1 in vitro. Growth Factors 5:149–158. 36. Guerne PA, Sublet A, Lotz M (1994) Growth factor responsiveness of human articular chondrocytes: distinct profiles in primary chondrocytes subcultured chondrocytes and fibroblasts. J Cell Physiol 158:476–484. 37. Boumediene K, Vivien D, Macro M, Bogdanowicz P, Lebrun E, Pujol JP (1995) Modulation of rabbit articular chondrocyte (RAC) proliferation by TGB-beta isoforms. Cell Prolif 28:221–234. 38. Parekh T, Saxena B, Reibman J, Cronstein BN, Gold LI (1994) Neutrophil migration in response to TGF-beta isoforms
(TGF-b1 TGF-b2 TGF-b3) is mediated by fibronectin. J Immunol 152:2456–2466. 39. Jacobsen SE, Keller JR, Ruscetti FW, Kondaiah P, Roberts AB, Falk LA (1991) Bidirectional effects of transforming growth factor beta (TGF-beta) on colony-stimulating factor induced human myelopoiesis in vitro: differential effects of distinct TGF-beta isoforms. Blood 78:2239–2247. 40. Merwin JR, Newman W, Beall LD, Tucker A, Madri J (1991) Vascular cells respond differently to transforming growth factors beta 1 and beta 2 in vitro. Am J Pathol 138:37–51. 41. Akhurst RJ, FitzPatrick DR, Fowlis DJ, Gatherer D, Millan FA, Slager H (1992) The role of TGFbs in mammalian development. Mol Reprod Dev 32:127–135. 42. Paulsson Y, Beckmann PM, Westermark B, Heldin C-H (1988) Density-dependent inhibition of cell growth by transforming growth factor-b1 in normal human fibroblasts. Growth Factors 1:19–27. 43. Fenderson BA, Stamenkovic I, Aruffo A (1993) Localisation of hyaluronan in mouse embryos during implantation gastrulation and organogenesis. Differentiation 54:85–98. 44. Turley EA (1992) Hyaluronan and cell locomotion. Cancer Metastasis Reviews 11:21–30. 45. Thomas L, Byers HR, Vink J, Stamenkovic I (1992) CD44H regulates tumor cell migration on hyaluronate coated substrata. J Cell Biol 118:971–977. 46. Samuel SK, Hurta RA, Spearman MA, Wright JA, Turley EA, Greenberg AH (1993) TGF-b1 stimulation of cell locomotion utilizes the hyaluronan receptor RHAMM and hyaluronan. J Cell Biol 123:749–758. 47. Ruiz P, Schwa¨rzler C, Gu¨nthert U (1995) CD44 isoforms during differentiation and development. BioEssays 17: 17–24. 48. Hall CL, Wang C, Lange LA, Turley EA (1994) Hyaluronan and the hyaluronan receptor RHAMM promote foacal adhesion turnover and transient tyrosine kinase activity. J Cell Biol 126:575–588. 49. Hall CL, Turley EA (1995) Hyaluronan-RHAMM mediated cell locomotion and signalling in tumorigenesis. J Neuro Oncol 26:221–229. 50. Schor SL (1980) Cell proliferation and migration within three-dimensional collagen gels. J Cell Sci 41:159–175. 51. McPherson JM, Piez KA (1988) Collagen in dermal wound repair In: Clark RAF, Henson PM eds, The Molecular and Cellular Biology of Wound Repair, Mew York: Plenum Press, pp. 471–496. 52. Mast BA, Diegelmann RF (1992) Scarless wound healing in the mammalian fetus. Surg Gynecol Obstet 174:441–451. 53. Adzick NS, Lorenz HP (1994) Cells matrix growth factors and the surgeon. Ann Surg 220:10–18. 54. Sullivan KM, Lorenz HP, Adzick NS (1993) The role of transforming growth factor beta in human fetal wound healing. Surg Forum 44:723–725. 55. Shah M, Foreman DM, Ferguson MWJ (1995) Neutralisation of TGF-b1 and TGF-b2 or exogenous addition of TGF-b3 to cutaneous rat wounds reduces scarring. J Cell Sci 108: 985–1002. 56. Krummel TM, Michna BA, Thomas BL, Sporn MB, Nelson JM, Salzberg AM, Cohen IK, Diegelmann RF (1988) Transforming growth factor beta (TGF-b) induces fibrosis in a fetal wound model. J Pediat Surg 23:647–652. 57. Ham RG (1980) Dermal fibroblasts In Harris C, Trump BF, Stoner G eds, Methods in Cell Physiology, Vol. 21, New York: Academic Press, pp. 255–276. 58. Schor SL, Court J (1979) Different mechanisms involved in the attachment of cells to native and denatured collagen. J Cell Sci 38:267–281. 59. Underhill CB, Toole BP (1979) Binding of hyaluronate to the surface of cultured cells. J Cell Biol 82:475–484.
Fetal and adult fibroblasts differ with respect to a number of phenotypic characteristics, including the synthesis of specific isoforms of matrix macromolecules,1 the production of cytokines2,3 and cellular response to these two classes of effector molecules.4–6 In this regard, we have recently reported that: (1) the migration of confluent fetal and adult fibroblasts is differentially affected by epidermal growth factor (EGF), transforming growth factor b1 (TGF-b1), and platelet-derived growth factor (PDGF), and (2) the synthesis of hyaluronan (HA) by fetal and adult fibroblasts is also differentially affected by these cytokines.7 The TGF-b family of cytokines are multifunctional regulators of various aspects of cell behaviour, including proliferation,8 matrix synthesis9 and cell migration.10–13 There are three principal mammalian
From the Department of Dental Surgery and Periodontology, The Dental School, University of Dundee, Dundee DD1 4HR, UK Correspondence to Seth Schor, E-mail: SLSchor.ITS.dundee.ac.uk Received 12 June 1997; accepted for publication 22 September 1997 7 1998 Academic Press Limited 1043–4666/98/040281 + 09 $25.00/0/ck970294 KEY WORDS: TGF-b/ fibroblasts/ migration/ hyaluronan/ wound healing CYTOKINE, Vol. 10, No. 4 (April), 1998: pp 281–289
isoforms of TGF-b.14 These commonly exert similar effects on cell behaviour,15 although several studies have revealed isoform-specific differences in both target cell responsiveness and mode of action.16 These observations are consistent with the differential expression of TGF-b isoforms during development17,18 and the resultant speculation that they may fulfill distinct functions in embryogenesis, as well as in the adult organism.19 Our previous work has indicated that the effect of TGF-b1 on fibroblast migration is dependent upon both developmental stage (fetal vs adult) and cell density.7,12,20 The objective of the present study has consequently been to extend these observations to include other members of the TGF-b family of cytokines.
RESULTS Fetal and adult fibroblasts were plated at subconfluent and confluent cell densities onto the surface of 3D collagen gel substrata in the absence (control) and presence of different concentrations of TGF-b1, TGF-b2, and TGF-b3. Data obtained with one representative fetal and one adult fibroblast line confirm our previous observations regarding the 281
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30 A
C
B
D
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20 10 0 15 10 5 0
0.1
1.0 10.0 0.1 1.0 Concentration TGF-β (ng/ml)
10.0
Figure 1. The effects of TGF-b isoforms on the migration of fetal (A,B) and adult (C,D) skin fibroblasts. Data are presented concerning the effects of TGF-b1 (q), TGF-b2 (<) and TGF-b3 (Q) on the migration of one fetal and one adult human skin fibroblast line. Cells were plated onto collagen gels at subconfluent (A,C) and confluent (B,D) cell densities and the effects of the different TGF-b isoforms on their subsequent migration into the three- dimensional gel matrix measured after a 4-day incubation period (as indicated in Materials and Methods). Data are expressed as the percentage of total cells present within the gel matrix (mean 2 SD of five replicate experiments). Dashed line indicates the percentage of cells present in the gel matrix of control cultures.
differential and density-dependent effects of TGF-b1 on these two cell populations (Fig. 1), i.e. TGF-b1 had no significant effect on the migration of subconfluent fetal fibroblasts, inhibited the migration of confluent fetal fibroblasts and subconfluent adult cells, and had no effect on confluent adult cells. Our data further TABLE 1.
Fetal
Adult
indicate that TGF-b2 exerts essentially the same effects as TGF-b1 on fibroblast migration, although there are quantitative differences with respect to the concentration of these isoforms required to achieve maximal inhibition of confluent fetal fibroblast migration. In contrast, TGF-b3 differed from the other two isoforms in that it inhibited the migration of subconfluent fetal fibroblasts and stimulated the migration of confluent adult fibroblasts. TGF-b3 did not differ from the -b1 and -b2 isoforms regarding its inhibitory effect on the migration of subconfluent adult and confluent fetal fibroblasts. Data summarizing the effects of TGF-b1, -b2 and -b3 on the migration of three fetal and three adult fibroblast lines are presented in Table 1 (expressed in terms of absolute migration) and Figure 2 (expressed in terms of migration relative to control). TGF-b isoforms were used at optimal concentrations, i.e. 10 ng/ml TGF-b1 and TGF-b2, and either 0.1 or 10 ng/ml TGF-b3. These data confirm that the motogenic activity of TGF-b1 and -b2 differ from that of TGF-b3 and are dependent upon both developmental stage of the target cells and their degree of confluence. The effects of TGF-b isoforms on HA synthesis by the same three fetal and adult fibroblast lines are summarized in Table 2 (absolute levels of [3 H]glucosamine incorporation) and Figure 3 (incorporation relative to control). These data parallel those relating to cell migration. Linear regression analysis (Fig. 4) of data obtained under each combination of cell type, cell density and TGF-b isoform confirms that there is a good correlation between cell migration and total HA synthesis under all experimental conditions
Effect of TGF-b isoforms on the migration of adult and fetal fibroblasts Control
TGF-b1
TGF-b2
TGF-b3
Subconfluent
F104 F105 FS6 Mean 2 SD
19.1 2 1.6 15.6 2 0.9 18.2 2 0.6 17.6 2 1.5
16.9 2 0.9 17.0 2 1.1 15.9 2 0.4 16.4 2 0.4
18.1 2 0.4 17.0 2 0.1 16.3 2 0.0 17.1 2 0.7
5.0 2 0.2 6.3 2 0.2 6.0 2 1.1 5.8 2 0.6
Confluent
F104 F105 FS6 Mean 2 SD
11.2 2 0.8 14.3 2 1.1 16.6 2 0.3 14.0 2 2.2
4.7 2 0.7 6.5 2 1.1 2.9 2 0.4 4.7 2 1.5
2.2 2 0.5 5.9 2 1.0 3.4 2 0.2 3.8 2 1.5
2.0 2 0.7 3.9 2 1.0 3.0 2 0.6 3.0 2 0.8
Subconfluent
FSF37 FSF44 SK319 Mean 2 SD
15.6 2 0.3 25.4 2 0.8 20.8 2 1.0 20.6 2 4.0
6.5 2 0.1 5.6 2 0.1 8.2 2 1.1 6.8 2 1.1
5.9 2 0.6 13.7 2 2.4 9.1 2 0.3 9.6 2 3.2
6.8 2 0.3 8.7 2 0.2 7.6 2 0.9 7.7 2 0.8
Confluent
FSF37 FSF44 SK319 Mean 2 SD
2.0 2 0.1 1.9 2 0.1 2.3 2 0.5 2.1 2 0.2
1.9 2 0.3 1.6 2 0.4 2.1 2 0.9 1.9 2 0.2
2.0 2 0.1 1.6 2 0.4 3.3 2 1.0 2.3 2 0.7
8.4 2 1.0 7.1 2 0.2 9.6 2 0.5 8.4 2 1.0
Data are presented concerning the effects of optimal concentrations of TGF-b1 (10 ng/ml), TGF-b2 (10 ng/ml) and TGF-b3 (0.1 or 10 ng/ml) on the migration of several adult and fetal fibroblast lines. Cell migration was determined microscopically on day 4, as described in Materials and Methods, and expressed as the percentage of cells within the gel matrix.
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1.5
1.5
Relative migration
A
Gel filtration chromatography was used to analyse the effects of TGF-b isoforms on the polydispersity of HA synthesized by representative fetal and adult fibroblast lines (Figs 5 and 6, respectively). These data indicate that the cell-density dependent effects of TGF-b isoforms on total HA synthesis (inhibitory, no effect, or stimulatory) result from a corresponding effect on the relative production of high molecular species of HA.
C
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4
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DISCUSSION
3
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2 0.5 1 0
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0 β3 β1 TGF-β isoforms
β2
β3
Figure 2. The effects of TGF-b isoforms on the migration of fetal (A,B) and adult (C,D) skin fibroblasts. Summary of data obtained with three lines of fetal and three lines of adult skin fibroblasts. Cells were plated at subconfluent (A,C) and confluent (B,D) densities and incubated with concentrations of the TGF-b isoforms which elicited maximal response (0.1 or 10 ng/ml TGF-b3 and 10 ng/ml TGF-b1 and TGF-b2), as indicated in Figure 1. Data are expressed as the percentage of cells within the gel matrix relative to control. Dashed line indicates control level of migration.
(r2 = 0.861; P Q 0.0001). The diverse effects of TGF-b isoforms on fetal and adult fibroblasts in terms of both cell migration and HA synthesis are summarized in Table 3. TABLE 2.
Fetal
Adult
The objective of this study has been to compare the response of fetal and adult skin fibroblasts to TGF-b isoforms with respect to cell migration and HA synthesis. Our results indicate that fetal and adult fibroblasts exhibit distinct responses to these isoforms, and that these differences are dependent upon: (1) isoform type; and (2) cell density.
Dependence on developmental state Fetal and adult fibroblasts cultured at identical cell densities responded differently to TGF-b isoforms in terms of both cell migration and HA synthesis. Our data indicate that: (1) the migration of subconfluent fetal cells was unaffected by TGF-b1 and -b2, but inhibited by TGF-b3, whilst the migration of subconfluent adult cells was inhibited by all three isoforms; and (2) the migration of confluent fetal cells was inhibited by all three TGF-b isoforms, whilst the migration of confluent adult cells was unaffected by TGF-b1 and -b2, but stimulated by TGF-b3. In the absence of exogenous effector molecules, control fetal
Effect of TGF-b isoforms on HA synthesis by adult and fetal fibroblasts Control
TGF-b1
TGF-b2
TGF-b3
Subconfluent
F104 F105 FS6 Mean 2 SD
123.2 2 3.5 136.7 2 2.9 103.9 2 2.1 121.3 2 13.4
118.1 2 10.0 140.6 2 9.6 100.6 2 3.1 119.8 2 16.4
126.5 2 12.6 125.6 2 5.2 101.5 2 9.3 117.9 2 11.6
78.6 2 4.2 95.6 2 1.9 80.6 2 2.5 84.9 2 7.6
Confluent
F104 F105 FS6 Mean 2 SD
120.1 2 12.9 141.7 2 6.3 113.9 2 6.6 125.2 2 11.9
58.9 2 14.3 80.6 2 6.9 59.9 2 26.7 66.5 2 10.0
14.5 2 8.5 60.1 2 5.6 64.7 2 8.4 46.3 2 22.7
53.7 2 23.2 58.5 2 7.5 62.1 2 9.6 58.1 2 3.4
Subconfluent
FSF37 FSF44 SK319 Mean 2 SD
114.0 2 1.9 104.3 2 2.8 100.1 2 2.6 106.2 2 5.9
15.7 2 0.6 11.5 2 2.9 16.0 2 1.9 14.4 2 2.0
18.7 2 3.6 6.6 2 4.5 19.5 2 3.6 14.9 2 5.9
17.0 2 0.5 25.8 2 1.5 11.3 2 0.9 18.0 2 6.0
Confluent
FSF37 FSF44 SK319 35.4 2 4.5
34.2 2 0.3 41.4 2 1.8 30.7 2 2.5 33.5 2 5.1
28.1 2 0.1 40.3 2 2.5 32.1 2 1.6 35.2 2 4.6
34.6 2 0.2 41.1 2 0.2 29.8 2 3.5 85.2 2 9.2
89.2 2 0.2 94.0 2 4.2 72.5 2 1.6
Mean 2 SD
Data are presented concerning the effects of optimal concentrations of TGF-b1 (10 ng/ml), TGF-b2 (10 ng/ml) and TGF-b3 (0.1 or 10 ng/ml) on HA by several adult and fetal fibroblast lines. HA synthesis was determined microscopically on day 4, as described in Materials and Methods, and expressed as dpm [3H]glucosamine incorporated in HA 10 − 5 cells × 103.
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1.5
1.5
4
C
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1.0
0.5
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0
0
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3
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Relative HA synthesis
A
3
2
1
D
B 1.0
2
0
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Figure 4.
0
0
The correlation between HA synthesis and cell migration of three fetal and adult lines in the absence and presence of TGF-b1, TGF-b2, and TGF-b3 were analysed by linear regression analysis (r 2 = 0.861; P Q 0.0001).
β1
β2
β1 β3 TGF-β isoforms
β2
β3
Figure 3. The effects of TGF-b isoforms on HA synthesis by fetal (A,B) and adult (C,D) skin fibroblasts. Summary of data obtained with three lines of fetal and three lines of adult skin fibroblasts. Cells were plated at subconfluent (A,C) and confluent (B,D) densities and incubated with concentrations of the TGF-b isoforms which elicited maximal response (0.1 or 10 ng/ml TGF-b3 and 10 ng/ml TGF-b1 and TGF-b2), as indicated in Figure 1. Data are expressed as [3H]glucosamine incorporated into HA relative to control. Dashed line indicates control level of HA synthesis.
and adult fibroblasts also differ with respect to their migratory behaviour and HA synthesis.21,22 These intrinsic differences appear to result from their differential production of motogenic cytokines2,3,24–27 and the modulation of HA synthesis by these effector molecules.7,12,28,29 Fetal and adult fibroblasts also differ with respect to a number of other biochemical characteristics of potential relevance to migration, including the activity of matrix synthesizing enzymes,30 the synthesis of specific isoforms of matrix glycoproteins1,31 and the secretion of matrix degrading enzymes.32,33
Dependence on TGF-b isoform TGF-b isoforms have commonly been found to elicit similar effects upon cell behaviour in vivo34 and in vitro,15,35–37 although there may be significant differences in their relative potencies.16 With specific reference to cell migration, Parekh et al.38 observed that TGF-b1, -b2 and -b3 all stimulated neutrophil migration in the transmembrane assay. Data presented in this communication indicate that the different TGF-b isoforms elicited distinct effects upon fibroblast migration and HA synthesis. Interestingly, TGF-b1 and -b2 were always similar in
1
2 3 Relative migration
4
5
Correlation between cell migration and HA synthesis.
their biological activity (either inhibitory or inactive); the bioactivity of TGF-b3 differed from that of the other two isoforms in subconfluent fetal fibroblasts (where it inhibited cell migration and HA synthesis) and in confluent adult cells (where it stimulated both cellular activities). These observations are consistent with previous reports documenting qualitative differences in the biological activity of TGF-b isoforms.39,40 In this regard, it is of interest that there are both temporal and spatial differences in the expression of the three TGF-b isoforms at both the protein and mRNA levels during embryonic development.17,18,41 Such differences suggest that TGF-b isoforms fulfil different functions and may continue to do so in the adult.19
Dependence on cell density The effects of all three TGF-b isoforms were modulated by cell density. Indeed, with the exception of the effect of TGF-b3 on fetal fibroblasts, all TGF-b isoforms elicited distinct effects at subconfluent and
TABLE 3. Effects of TGF-b isoforms on fetal and adult fibroblasts Fetal
Adult
Subconfluent
TGF-b1 TGF-b2 TGF-b3
t t 4
4 4 4
Confluent
TGF-b1 TGF-b2 TGF-b3
4 4 4
t t 3
Data summarizing the effects of TGF-b isoforms on both cell migration and HA synthesis. Key: 4, inhibition; t, no effect; 3, stimulation.
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Vt
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25
0
75
50
25
0
75
50
25
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50 0 10 Fraction number
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30
40
50
Figure 5. Effect of TGF-b isoforms on the size class distribution of HA synthesized by subconfluent (A,B,C,D) and confluent (E,F,G,H) fetal fibroblasts. Cells were incubated with concentrations of TGF-b isoforms which elicited maximal response, as indicated in Fig. 1. The polydispersity of HA synthesized by fetal fibroblasts plated on the surface of a 3D collagen gel was analysed using Sepharose CL-2B column chromatography as described in the Materials and Methods. Vdb indicates the point at which dextran blue was eluted from the column and is an estimate of the void volume; Vt indicates the total column volume.
confluent cell densities (summarized in Table 3). The inhibition of fibroblast proliferation by TGF-b1 has previously been reported to be modulated by cell density.42 Cell density also affects the behaviour of control fetal and adult fibroblasts with respect to their migratory activity21 and HA synthesis.22 The densitydependent effects of the different TGF-b isoforms on
fetal and adult cells reported here presumably reflect the differential response of cells in different physiological states with respect to their intrinsic level of cell migration and HA synthesis, as well as other potentially relevant parameters including the synthesis of cytokines and matrix molecules, their respective cell surface receptors and matrix degrading enzymes.
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Figure 6. Effect of TGF-b isoforms on the size class distribution of HA synthesized by subconfluent (A,B,C,D) and confluent (E,F,G,H) adult fibroblasts. Cells were incubated with concentrations of TGF-b isoforms which elicited maximal response, as indicated in Fig. 1. The polydispersity of HA synthesized by adult fibroblasts plated on the surface of a 3D collagen gel was analysed using Sepharose CL-2B column chromatography as described in the Materials and Methods. Vdb indicates the point at which dextran blue was eluted from the column and is an estimate of the void volume; Vt indicates the total column volume. (A,E), control; (B,F), TGF-b1; (C,G), TGF-b2; (D,H), TGF-b3.
Correlation between HA synthesis and cell migration: implications for cell behaviour during development and in the adult Data presented in Figure 4 indicate a close correlation between cytokine-induced alterations in cell migration and HA synthesis. A similar correlation has previously been observed with a number of other motogenic cytokines.7 In this regard it is of particular interest that there is a significant spatial and temporal correlation between TGF-b expression and HA
concentration during embryonic development;43 these observations led the authors to speculate that TGF-b modulates HA synthesis and that this alteration in matrix may in turn mediate many of the biological effects of TGF-b. This postulated mechanistic link is consistent with the reported effect of HA on the migration of a number of cell types.44 These effects of HA on cell migration are mediated by interaction with its receptors, CD-44 and RHAMM.45,46 CD-44 is a complex family of related molecules produced by alternative splicing.47 Individual CD-44 isoforms
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display developmentally regulated differences in both cell expression and ligand affinity. Signal transduction elicited by ligation of RHAMM which may affect cell migration include tyrosine phosphorylation of the focal adhesion kinase pp125FAK.48,49 Related studies have indicated that the effects of several cytokines on cell migration may be both mediated and modulated by the extracellular matrix.38,50 All three TGF-b isoforms appeared to affect the synthesis of high molecular mass HA. A similar preferential modulation of high molecular mass HA has been reported for a number of cytokines, including TGF-b1.7 Our previous data suggest that alterations in this particular size class of HA are primarily responsible for HA-mediated changes in cell motility.7,12,20 The differential effects of TGF-b isoforms -b1, -b2 and -b3 on the migration of fetal and adult fibroblasts reported here may be relevant to differences in fetal and adult wound healing and the manner in which this is affected by members of the TGF-b family. Adult wound healing is a ‘‘reparative’’ process, commonly accompanied by scar formation;51 in contrast, dermal wound healing in the early gestation fetus is regenerative in nature and essentially scar-free.52,53 TGF-b1 and -b2 have been detected in adult wounds, whilst TGF-b3 is the principal isoform found in fetal wounds.54 Treatment of adult wounds with either neutralising antibodies for TGF-b1 and -b2, or exogenous application of TGF-b3, significantly reduced scar formation in healing adult wounds;55 conversely, application of TGF-b1 to fetal wounds induced excessive scar formation.56
MATERIALS AND METHODS Reagents Human platelet-derived TGF-b1, TGF-b2, and human recombinant TGF-b3 was either a generous gift from Dr A. Roberts (NIH, MD) or purchased from R&D Systems, Ltd. (Cowley, Oxford, UK). -[3 H]glucosamine hydrochloride (22 Ci/mmol) was obtained from Amersham International plc (Amersham, Bucks., UK). Hyaluronan (type 1, sodium salt), chondroitin-6-sulfate and hyaluronate lyase were obtained from Sigma Chemical Co. (Poole, Dorset, UK). Ultragold scintillation fluid was obtained from Canberra Packard (Pangbourne, Berkshire, UK). Eagle’s Minimal Essential Medium (MEM), donor calf serum, sodium pyruvate, glutamine, non-essential amino acids, antibiotics and tissue culture plastic dishes were obtained from Gibco BioCult (Paisley, UK). All other chemicals were obtained from BDH Chemicals (Poole, Dorset, UK) or Sigma Chemical Co. (Poole, Dorset, UK).
Cell culture and preparation of collagen gels The fetal and adult fibroblast lines used in this study were established in our laboratory by explant culture from
skin biopsies.57 Stock cultures were maintained in Eagle’s MEM supplemented with 15% (V/V) donor calf serum, 1 mM sodium pyruvate, 2 mM glutamine, non-essential amino acids at 37°C in a moist atmosphere containing 5% CO2. Cells were grown on 90 mm plastic tissue culture Petri dishes and passaged at a split ratio of 1:5 upon reaching confluence 7–10 days after plating. All experiments were performed with fibroblasts between passage 10–18. Cultures used in this study were shown to be free of mycoplasma contamination by staining with Hoechst 33256. Type I collagen was extracted from rat tail tendons in 3% acetic acid, dialysed for 2 days against distilled water, diluted to 2 mg/ml and used to make 2 ml collagen gels in 35 mm plastic tissue culture dishes as previously described.58
Migration assay Collagen gels were overlaid with 1 ml of either SF-MEM (controls) or serum-free MEM containing 4× the final concentration of cytokine. Confluent stock cultures were trypsinized, pelleted by centrifugation, resuspended in growth medium containing 20% donor calf serum at a concentration of either 2.5 × 104 cells/ml (subconfluent) or 2.5 × 105 cells/ml (confluent) and 1-ml aliquots of this inoculum were pipetted onto the cultures. Considering the 2-ml volume of the collagen gel, the 1-ml medium overlay and the 1-ml cell inoculum, this procedure gives a final concentration of 5% serum in both control and cytokinecontaining cultures. The cultures were incubated for 4 days and the percentage of fibroblasts found within the 3D gel matrix was then ascertained by microscopic observation of 15–20 randomly selected fields, as previously described.50 Replicate gels were counted for each experimental condition.
Metabolic labelling and determination of total labelled HA Glycosaminoglycans (GAGs) synthesized by the fibroblasts were metabolically labelled by incubating cultures with 2.5 mCi/ml of [3 H]glucosamine. These cultures were separated into medium and pericellular fractions, as previously described by Ellis et al.12 In this protocol, the medium and gel were transferred to a centrifuge tube and spun at 1000 × g for 20 min at 4°C. The medium was removed and the compacted gel was resuspended in 2 ml of phosphatebuffered saline. Centrifugation was repeated, and the supernatant was combined with the previous one to form the medium fraction. The pericellular fraction was obtained by extracting the cell layer with 2 ml 50 mM Tris–HCl, 4 M guanidinium chloride, pH 7.4, for 24 h at 4°C. The insoluble material was removed by centrifugation at 12 000 × g for 2 min in a microcentrifuge and the supernatant was collected as the pericellular fraction. The medium and pericellular fractions were dialysed against 0.1 M sodium acetate, 5 mM EDTA, pH 5.5 for 48 h (with two changes per day). The dialysate volumes were measured and six 200-ml aliquots were taken of each sample. 20 ml of Streptomyces sp. hyaluronidase (HA lyase: EC 4.2.2.1), at a final concentration 0.5 U/ml in acetate buffer, was added to three of these, whilst 20 ml of acetate buffer was added to the remaining three
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controls. All samples were incubated for 17 h at 37°C, then boiled for 1 min to inactivate the enzyme and unlabelled carrier GAG added (30 ml 1% chondroitin sulfate/0.5% hyaluronate in PBS). The undigested GAGs were precipitated with 4 volumes of 1.3% potassium acetate in 100% ethanol at −20°C for 3 h.59 The precipitate was sedimented by centrifugation at 12 000 × g for 5 min, the supernatant removed and the pellet washed in 1 ml 75% ethanol and sedimented as above. The supernatant was again removed and the pellet redissolved in 100 ml of 1 M sodium hydroxide and boiled for 3 min. The sodium hydroxide was neutralized with 100 ml of 1 M hydrochloric acid and radioactivity determined using a Packard scintillation counter. The activity (dpm) associated with HA was taken as the difference between the counts in the control and enzyme digested preparations. We consistently found that less than 5% of the total synthesized HA was present in the pericellular fraction under all experimental conditions; data are consequently only presented for HA present in the medium fraction. We have previously confirmed the reliability of estimating HA synthesis by radiolabel incorporation by demonstrating the close correlation between the results obtained by this method and biochemical determination of chemical mass.22
Molecular size distribution of labelled HA The molecular size distribution of labelled material was analysed by Sepharose CL-2B gel filtration chromatography of 200-ml aliquots; columns had an internal diameter of 10 mm, contained 25 ml of gel and were equilibrated with 50 mM Tris–HCl, 4 M guanidinium chloride, pH 7.4. Samples of labelled medium and pericellular fractions were first exhaustively dialysed against 50 mM sodium acetate, 10 mM NaCl, pH 5.5. Representative aliquots were digested prior to chromatography with 2 U of Streptomyces sp. hyaluronidase for 17 h in order to identify the radiolabelled HA. The column was eluted with Tris/guanidinium hydrochloride buffer at 6 ml/h. Fractions (0.5 ml) were collected and radioactivity determined with a Packard scintillation counter. VDB and Vt were determined by the elution of dextran blue and sodium dichromate, respectively. The relative proportions of high (empirically defined as q2 × 106 kDa) and low molecular weight HA were estimated by cutting out and weighing the peaks in the radioactivity profile associated with the void (estimated by VDB) and included volumes, respectively.
Statistical analysis All statistical tests were performed using Prism 2 graphics software (GraphPad Software, San Diego, California). The two-tailed t-test was employed.
Acknowledgements This work has been supported by a grant from the Wellcome Trust.
CYTOKINE, Vol. 10, No. 4 (April, 1998: 281–289)
REFERENCES 1. Matsura H, Hakomori SI (1985) The oncofetal domain of fibronectin defined by monoclonal antibody FDC-6 its presence in fibronectin from fetal and tumor tissues and its absence from normal adult tissues and plasma. Proc Natl Acad Sci USA 82:6517–6521. 2. Grey AM, Schor AM, Rushton G, Ellis I, Schor SL (1989) Purification of the migration stimulating factor produced by fetal and breast cancer patient fibroblasts. Proc Natl Acad Sci USA 86:2438–2442. 3. Stoker M, Gherardi E, Perryman M, Gray J (1987) Scatter factor is a fibroblast-derived modulator of epithelial cell mobility. Nature 327:239–242. 4. Nathan C, Sporn M (1991) Cytokines in context. J Cell Biol 113:981–986. 5. Schor SL (1994) Cytokine control of cell motility: modulation and mediation by the extracellular matrix. Prog Growth Factor Res 5:223–248. 6. Schor SL, Ellis I, Banyard J, Dolman C, Seneviratne K, Gilbert AD, Chisholm DM (1996) Fetal-like phenotypic characteristics of gingival fibroblasts: Potential relevance to wound healing. Oral Dis 2:155–166. 7. Ellis I, Banyard J, Schor SL (1997) Differential response of fetal and adult fibroblasts to cytokines: cell migration and hyaluronan synthesis. Development 124:1593–1600. 8. Roberts AB, Anzano MA, Wakefield LM, Roche NS, Stern DF, Sporn MB (1985) Typeb transforming growth factor: A bifunctional regulator of cellular growth. Proc Natl Acad Sci USA 82:119–123. 9. Roberts AB, Heine UI, Flanders KC, Sporn MB (1990a) Transforming growth factor-b: Major role in regulation of extracellular matrix. Ann New York Acad Sci 580:225–232. 10. Wahl SM, Hunt DA, Wakefield LM, McCartney-Francis N, Wahl LM, Roberts AB, Sporn MB (1987) Transforming growth factor b induces monocyte chemotaxis and growth factor production. Proc Natl Acad Sci USA 84:5788–5792. 11. Postlethwaite AE, Keski-Oja J, Moses HL, Kang AH (1987) Stimulation of chemotactic migration of human fibroblasts by TGF-b. J Exp Med 165:251–256. 12. Ellis I, Grey AM, Schor AM, Schor SL (1992) Antagonistic effects of TGFb-1 and MSF on fibroblast migration and hyaluronic acid synthesis: Possible implications for dermal wound healing. J Cell Sci 102:447–456. 13. Adelmann-Grill BC, Wach F, Cully Z, Hein R, Krieg T (1990) Chemotactic migration of normal dermal fibroblasts towards EGF and its modulation by PDGF and TGF-b. Eur J Cell Biol 51:322–326. 14. Massague J (1990) The transforming growth factor b family. Annu Rev Cell Biol 6:597–641. 15. Graycar JL, Miller DA, Arrick BA, Lyons RM, Moses HL,Derynck R (1989) Human transforming growth factor-b3: Recombinant expression purification and biological activities in comparison with transforming growth factors-b1 and -b2. Mol Endocrinol 3:1977–1986. 16. Roberts AB, Kondaiah P, Rosa F, Watanabe S, Good P, Danielpour D, Roche NS, Rebbert ML, Dawid IB, Sporn MB (1990) Medoderm induction in Xenopus laevis distinguishes between the various TGFb isoforms. Growth Factors 3:277–286. 17. Pelton RW, Dickson ME, Moses HL, Hogan BLM (1990) In situ hybridization of TGF-b3 RNA expression during mouse development: comparative studies with TGF-b1 and -b2. Development 110:609–620. 18. Akhurst RJ, FitzPatrick DR, Gatherer D, Lehnert SA, Millan FA (1990) Transforming growth factor betas in mammalian embryogenesis. Prog Growth Factor Res 2:153–168. 19. Roberts AB, Sporn MB (1992) Differential expression of TGF-beta isoforms in embryogenesis suggests specific roles in developing and adult tissues. Mol Reprod Dev 32:91–98.
(p.b.e.) — CYTO — 10/4 — PRESS SET — ( 847375 ) —MS 0199 Distinct effect of TGF-b isoforms / 289 20. Ellis IR, Schor SL (1996) Differential effects of TGF-b1 on hyaluronan synthesis by fetal and adult skin fibroblasts: Implications for cell migration and wound healing. Exp Cell Res 228:326–333. 21. Schor SL, Schor AM, Rushton G, Smith L (1985) Adult fetal and transformed fibroblasts display different migratory phenotypes on collagen gels: evidence for an isoformic transition during fetal development. J Cell Sci 73:221–234. 22. Chen J, Grant ME, Schor AM, Schor SL (1989) Differences between adult and foetal fibroblasts in the regulation of hyaluronate synthesis: correlation with migratory activity. J Cell Sci 94:577–589. 23. Clemmons DR (1983) Age dependent production of a competence factor by human fibroblasts. J Cell Physiol 114: 61–67. 24. Schor SL, Schor AM, Grey AM, Rushton G (1988) Fetal and cancer patient fibroblasts produce an autocrine migration stimulating factor not made by normal adult cells. J Cell Sci 90:391–399. 25. Kondo H, Matsuda R, Yonezawa Y (1993) Autonomous migration of human fetal skin fibroblasts into a denuded area in a cell monolayer is mediated by basic fibroblast growth factor and collagen. In Vitro Cellul Dev-Animal 29A:929–935. 26. Kondo H, Yonezawa Y (1995) Fetal-adult transition in terms of their serum dependency and growth factor requirements of human skin fibroblast migration. Exp Cell Res 220:501–504. 27. Locci P, Lilli C, Marinucci L, Baroni T, Pezzetti F, Becchetti E (1993) Embryonic skin fibroblasts release TGF-alpha and TGF-beta able to influence synthesis and secretion of GAG. Cell Mol Biol 39:415–426. 28. Schor SL, Schor AM, Grey AM, Chen J, Rushton G, Grant ME, Ellis I (1989) Mechanism of action of the migration stimulating factor (MSF) produced by fetal and cancer patient fibroblasts: effect on hyaluronic acid synthesis. In Vitro 25:737–746. 29. Heldin P, Laurent TC, Heldin C-H (1989) Effect of growth factors on hyaluronan synthesis in cultured human fibroblasts. Biochem J 258:919–922. 30. Duncan BW, Qian J, Liu X, Bhatnagar R (1992) Regulation of prolyl hydroxylase activity in fetal and adult fibroblasts In: Adzick NS, Longaker MT eds, Fetal wound healing, New York: Elsevier Scientific Press, pp. 303–323. 31. ffrench-Constant C, Van der Water L, Dvorak HF, Hynes RO (1989) Reappearance of embryonic pattern of fibronectin splicing during wound healing in the adult rat. J Cell Biol 109:903–914. 32. Sottile J, Mann DM, Diemar V, Mills AJT (1989) Regulation of collagenase and collagenase mRNA production by early and late passage human diploid fibroblasts. J Cell Physiol 138:281–290. 33. Basset AH, Bellocq JP, Wolf C, Stoll I, Hutin P, Limacher JM, Podhajcer OL, Chenard MP, Rio MC, Chambon P (1990) A novel metalloproteinase gene specifically expressed in stromal cells of breast carcinomas. Nature 348:699–704. 34. Daniel CW, Robinson SD (1992) Regulation of mammary growth and function by TGF-beta. Mol Reprod Dev 32:145–151. 35. Merwin JR, Roberts A, Kondaiah P, Tucker A, Madri J (1991) Vascular cell responses to TGF-beta 3 mimic TGF-beta 1 in vitro. Growth Factors 5:149–158. 36. Guerne PA, Sublet A, Lotz M (1994) Growth factor responsiveness of human articular chondrocytes: distinct profiles in primary chondrocytes subcultured chondrocytes and fibroblasts. J Cell Physiol 158:476–484. 37. Boumediene K, Vivien D, Macro M, Bogdanowicz P, Lebrun E, Pujol JP (1995) Modulation of rabbit articular chondrocyte (RAC) proliferation by TGB-beta isoforms. Cell Prolif 28:221–234. 38. Parekh T, Saxena B, Reibman J, Cronstein BN, Gold LI (1994) Neutrophil migration in response to TGF-beta isoforms
(TGF-b1 TGF-b2 TGF-b3) is mediated by fibronectin. J Immunol 152:2456–2466. 39. Jacobsen SE, Keller JR, Ruscetti FW, Kondaiah P, Roberts AB, Falk LA (1991) Bidirectional effects of transforming growth factor beta (TGF-beta) on colony-stimulating factor induced human myelopoiesis in vitro: differential effects of distinct TGF-beta isoforms. Blood 78:2239–2247. 40. Merwin JR, Newman W, Beall LD, Tucker A, Madri J (1991) Vascular cells respond differently to transforming growth factors beta 1 and beta 2 in vitro. Am J Pathol 138:37–51. 41. Akhurst RJ, FitzPatrick DR, Fowlis DJ, Gatherer D, Millan FA, Slager H (1992) The role of TGFbs in mammalian development. Mol Reprod Dev 32:127–135. 42. Paulsson Y, Beckmann PM, Westermark B, Heldin C-H (1988) Density-dependent inhibition of cell growth by transforming growth factor-b1 in normal human fibroblasts. Growth Factors 1:19–27. 43. Fenderson BA, Stamenkovic I, Aruffo A (1993) Localisation of hyaluronan in mouse embryos during implantation gastrulation and organogenesis. Differentiation 54:85–98. 44. Turley EA (1992) Hyaluronan and cell locomotion. Cancer Metastasis Reviews 11:21–30. 45. Thomas L, Byers HR, Vink J, Stamenkovic I (1992) CD44H regulates tumor cell migration on hyaluronate coated substrata. J Cell Biol 118:971–977. 46. Samuel SK, Hurta RA, Spearman MA, Wright JA, Turley EA, Greenberg AH (1993) TGF-b1 stimulation of cell locomotion utilizes the hyaluronan receptor RHAMM and hyaluronan. J Cell Biol 123:749–758. 47. Ruiz P, Schwa¨rzler C, Gu¨nthert U (1995) CD44 isoforms during differentiation and development. BioEssays 17: 17–24. 48. Hall CL, Wang C, Lange LA, Turley EA (1994) Hyaluronan and the hyaluronan receptor RHAMM promote foacal adhesion turnover and transient tyrosine kinase activity. J Cell Biol 126:575–588. 49. Hall CL, Turley EA (1995) Hyaluronan-RHAMM mediated cell locomotion and signalling in tumorigenesis. J Neuro Oncol 26:221–229. 50. Schor SL (1980) Cell proliferation and migration within three-dimensional collagen gels. J Cell Sci 41:159–175. 51. McPherson JM, Piez KA (1988) Collagen in dermal wound repair In: Clark RAF, Henson PM eds, The Molecular and Cellular Biology of Wound Repair, Mew York: Plenum Press, pp. 471–496. 52. Mast BA, Diegelmann RF (1992) Scarless wound healing in the mammalian fetus. Surg Gynecol Obstet 174:441–451. 53. Adzick NS, Lorenz HP (1994) Cells matrix growth factors and the surgeon. Ann Surg 220:10–18. 54. Sullivan KM, Lorenz HP, Adzick NS (1993) The role of transforming growth factor beta in human fetal wound healing. Surg Forum 44:723–725. 55. Shah M, Foreman DM, Ferguson MWJ (1995) Neutralisation of TGF-b1 and TGF-b2 or exogenous addition of TGF-b3 to cutaneous rat wounds reduces scarring. J Cell Sci 108: 985–1002. 56. Krummel TM, Michna BA, Thomas BL, Sporn MB, Nelson JM, Salzberg AM, Cohen IK, Diegelmann RF (1988) Transforming growth factor beta (TGF-b) induces fibrosis in a fetal wound model. J Pediat Surg 23:647–652. 57. Ham RG (1980) Dermal fibroblasts In Harris C, Trump BF, Stoner G eds, Methods in Cell Physiology, Vol. 21, New York: Academic Press, pp. 255–276. 58. Schor SL, Court J (1979) Different mechanisms involved in the attachment of cells to native and denatured collagen. J Cell Sci 38:267–281. 59. Underhill CB, Toole BP (1979) Binding of hyaluronate to the surface of cultured cells. J Cell Biol 82:475–484.