Alteration of the extracellular matrix of cultured human keratinocytes by transformation and during differentiation

Abstract

We have investigated the production of 3 extracellular matrix proteins (fibronectin, laminin and entactin) and glycosaminoglycans (GAGs) by keratinocytes derived from human squamous-cell carcinomas (SCCs). All the SCC lines retained the ability to synthesize fibronectin, laminin and entactin, and to incorporate them into an extracellular matrix. In some of the SCC lines fibronectin production was higher than in normal keratinocytes, and in most lines laminin production was equal to or higher than that seen in normal keratinocyte strains. All the tumorigenic SCC lines produced less entactin than normal keratinocytes. Five out of 7 SCC lines showed a shift in GAG production compared with normal keratinocytes, so that in these lines heparan sulphate was the major GAG as opposed to hyaluronic acid in the normal keratinocytes. As these changes could have been a consequence of the reduced differentiation found in the SCC lines, we fractionated normal keratinocytes into 4 size classes corresponding to different stages of differentiation, using Percoll gradient centrifugation. It was found that the ability to produce these extracellular matrix glycoproteins and GAGs was lost as keratinocytes terminally differentiated. However, this did not explain the matrix changes seen in the SCC lines, since the undifferentiated normal keratinocytes produced a normal pattern of extracellular matrix components. Nonetheless, the loss of extracellular matrix production may well explain the reduction in substratum adhesiveness which occurs as keratinocytes terminally differentiate.

Sulphation and fibronectin-binding properties of heparan sulphate glycosaminoglycans from transformed cultured human keratinocytes

“…Thus an alteration in the overall level of heparan sulphate sulphation is almost certainly not a factor in the ECM alterations which are observed in transformed keratinocytes (Brown & Parkinson, 1984;; although we cannot rule out the possibility that changes in the distribution of ester (o) sulphate groups may have occurred, since this would not have been detected by the methods employed here. Furthermore, these results contrast the many other reports of undersulphated heparan sulphates being produced by other types of transformed cells (Underhill & Keller, 1975;Winterbourne & Mora, 1981;Stamatoglou & Keller, 1983;David & van den Berghe, 1983;Robinson et al, 1984).…”

“…One important factor in this loss of ECM is probably a decrease in the sulphation of heparan sulphate, which has been reported in several systems (Underhill & Keller, 1975;Winterbourne & Mora, 1981; Stamatoglou & Keller, 1983;David & van den Berghe, 1983;Robinson et al, 1984). This decrease in sulphation reduces the affinity of heparan sulphate for fibronectin (Stamatoglou & Keller, 1983;Robinson et al, 1984) and may therefore interfere with the complex interactions between the various glycoprotein and proteoglycan components of the ECM, in which heparan sulphate is thought to play a central role (Gallagher et al, 1986 (Rheinwald, 1980), as previously described (Brown & Parkinson, 1983). The cells used in this study were: normal strains Z and R (derived from newborn foreskin and the foreskin of a 10-year-old respectively); SV6-1 Bam/HFK, an SV40 transformed keratinocyte cell line (Brown & Parkinson, 1984;Brown & Gallimore, manuscript (Rheinwald & Beckett, 1981).…”

“…Electrophoretic separations have previously shown that identical GAG preparations from normal keratinocytes are composed of hyaluronic acid (-.60%), heparan sulphate (-.30%) and chondroitin sulphates (-10%) (Brown & Parkinson, 1983). Therefore, by comparison with the electrophoresis results the peaks from the DEAE column were preliminarily identified as hyaluronic acid (peak I), heparan sulphate (peak II) and chondroitin sulphate (peak III).…”

“…Nitrous acid treatment was by the method of Wusteman (1979), as described previously (Brown & Parkinson, 1983).…”

“…Although SCC27 has only small amounts of fibronectin in its ECM, it also only secretes small amounts of fibronectin into the culture medium (Brown & Parkinson, 1985); and so the low amounts of fibronectin in its ECM could be due to low levels of synthesis, rather than a failure in ECM assembly due to the production of an undersulphated heparan sulphate. SCC27 shows no other obvious distinguishing features from the other SCC lines (Brown & Parkinson, 1985), which strongly suggests the production of an undersulphated heparan sulphate is not essential for either the expression of a transformed phenotype or for tumorigenicity in the keratinocyte. Furthermore, since our SV40 transformed line SV6-1 Bam/HFK produces invasive SCCs in nude mice (at high passage levels, Brown & Gallimore, manuscript in preparation), these results imply that even malignant transformation in the keratinocyte does not require a decrease in heparan sulphate sulphation.…”