CONCLUSIONES, RECOMENDACIONES Y SUGERENCIAS PARA

In the previous section, KGF was found to be up-regulated by CsA, NIF and PHT in

vitro, although the mechanism involved in regulation of this activity of these drugs is not

known. Nevertheless, it has been reported that these drugs are involved in the synthesis of certain bioactive molecules, including growth factors and cytokines, which further regulates various activities of cells (Brown et al. 1991; Seymour et al. 1996). PDGF, EGF, TGF p, lL-1, lL-6 are among these bioactive molecules. Notably these molecules have also been recognized as potent inducers of KGF in fibroblast cell lines originated from human and murine species (Brauchle et al. 1994; Chedid et al. 1994). Thus it is possible that the drugs might induce KGF through these molecules. In order to elucidate the mechanism involved in increased secretion o f KGF in drug-treated gingival fibroblasts, the effects of serum, PDGF, lL-1 and DHT on KGF secretion were measured in gingival fibroblast supernatants. Control cultures were incubated with low serum (1%) containing full culture media.

3.4.1 Effects of Serum on KGF

Although serum is known to be a potent inducer of KGF in dermal fibroblasts and murine 3T3 cells, its effect on gingival fibroblasts is not yet known. Since fibroblasts derived from different sources respond differently to the same stimulus (Hassell et al. 1976), this part of the study was carried out to examine the effects of serum on KGF secretion by normal gingival fibroblasts.

After serum starvation, gingival fibroblasts (n = 3) were incubated with full culture media containing 10% FCS, as described in section 2.4.2 and KGF measured in the supernatants twice using ELISA (section 2.2.3.2). The results showed that KGF secretion increased

with time both in control and serum-stimulated cultures, however a relatively higher amount being found in the latter. The results of 6 separate assays carried out with serum- stimulated gingival fibroblast supernatants are summarised in Table 3.6. The maximum relative level of KGF in serum-stimulated cultures was observed on day 3 and was 2.35 times higher than the amount of KGF present in the controls (0.39 ±0.16 ng/ml; range 0.17 to 0.58).

Table 3.6. Effects of growth mediators on KGF secretion by gingival fibroblasts in vitro

Conditions

KGF

Relative level (± SEM) Range (ng/ml)

p value

Serum 2.35 ±0.31 0.22 -1.66 0.003

PDGF 2.82 ±0.72 0.30-1.06 0.02

IL-1 2.58 ±0.14 0.49-2.17 0.003

DHT 1.72 ±0.20 0.35 -1.84 0.002

The values shown are the relative level of KGF secretion ± SEM in indicated condition. Range of KGF is shown.

The non-conditioned media containing 1 and 10% FCS were also subjected to ELISA to determine the indigenous KGF, to confirm that KGF detected in the culture supernatants (conditioned media) was secreted by the cells, and did not represent the innate KGF. The results of 2 separate experiments, carried out in triplicate, showed that average KGF in

10% FCS-containing medium was 0.07 ng/ml, whereas KGF was not detected in 1% FCS-containing medium.

In order to determine whether the elevated KGF induced by serum in cultures correlated with increased KGF gene transcription, semi-quantitative PCR was carried out using GAPDH as the internal control and the relative level o f KGF mRNA was measured, as described in section 2.6.2.4. The results showed an increase in KGF gene transcription ^with time, attaining a maximum KEI at 72 h of serum stimulation. Mean KEI obtained from 3 separate experiments in serum-stimulated cultures at 72 h was found to be 2.1 (± 0.54) times greater than the basal level (average KEI 19 ± 2.27; range 16 - 22).

3.4.2 Effects of PDGF BB on KGF secretion

The results in section 3.4.1 showed up-regulation of KGF in serum-stimulated cultures both at the protein and transcription levels. In order to identify the serum components responsible for the stimulatory effect on KGF expression, gingival fibroblasts (n = 3) were treated with 10 ng/ml of PDGF (section 2.4.2) and KGF was measured in the supernatants using ELISA. As shown in Table 3.6, a maximum relative level of KGF secretion induced by PDGF was measured on day 3 and was 2,82 fold greater than the control (average 0.27 ±0.10; range 0.17 to 0.44 ng/ml).

3.4.3 Effects of IL-1 on KGF secretion

IL-1 has been shown to be involved in the molecular pathogenesis o f GH (Myrillas et al.

1999) and it is also a potent inducer of KGF (Chedid et al. 1994). It is possible therefore that CsA, NIF and PHT induce KGF through IL-1. To examine this, normal gingival fibroblasts (n = 4) were treated with IL-1 (both a and p) in 1% FCS containing culture media, as described in section 2.4.2 and KGF was measured in the supernatants using ELISA assay, as described in section 2.2.3.2. The result showed that KGF secretion increased with time in control as well as in IL-1 treated cultures. A maximum relative level of KGF secretion was observed on day 4 with both isoforms of IL-1, which was on average 2.58 times greater than in the control cultures (average 0.62 ± 0.22; range 0.21 to 1.54 ng/ml), as shown in Table 3.6.

3.4.4 Effects of testosterone on KGF secretion

A possible role of testosterone in the pathogenesis of GH was shown by Dayan et al.

(1998), although the exact mechanism is unknown. Evidence suggests that KGF functions as a mediator of testosterone (Peehl and Rubin 1995) and is also elevated in prostate stromal cells in response to DHT (Planz et a l 1998). However, the effect of testosterone on KGF secretion by gingival fibroblasts has not yet been evaluated. This part of the study therefore evaluated the effect of testosterone on KGF secretion by normal gingival fibroblasts. The cells (n = 4) were serum starved for 48 h and treated with 20 ng/ml DHT in phenol-red free medium containing charcoal treated serum and KGF was measured in the supernatants using ELISA. As shown in Table 3.6 a maximum relative level o f KGF secretion in DHT-treated cultures was observed on day 3, and was

Thus, this section of the study show that KGF secretion is elevated by PDGF, IL-1 and testosterone, which are known to be inducers of KGF in fibroblast cell lines originated from other tissues or organs.