Long-term cyclosporin A exposure suppresses cathepsin-B and -L activity in gingival fibroblasts.

BACKGROUND: Gingival overgrowth is a common side-effect following administration of cyclosporin A. We reported previously that lysosomal protease cathepsin-L activity, but not cathepsin-B, was significantly suppressed by short-term cyclosporin A exposure in human gingival fibroblasts. Although this suppression may lead to decreased degradation of gingival connective tissue, a net increase in matrix proteins, and gingival overgrowth, the effects of cyclosporin A need to be more elucidated, considering the long-term use for patients following organ transplantation. OBJECTIVE: The aim of the present study was to evaluate the effects of clinically relevant doses of cyclosporin A on cultured human gingival fibroblasts. We evaluated the effects of long-term cyclosporin A exposure on cell proliferation, mRNA expression of various proteases and both cathepsin-B and -L activity in human gingival fibroblasts. MATERIALS AND METHODS: Human gingival fibroblasts were isolated from three donors' healthy gingiva and cultured from five to eight passages with or without 200 ng/ml of cyclosporin A. Proliferative activity of cyclosporin A-treated cells was examined using MTT assay. Total RNA and cellular proteins were collected for semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis and for measurement of the cathepsin-B and -L activity. RESULTS: Long-term cyclosporin A exposure had no effects on cell proliferation. Accumulation of cathepsin-B, -H and -L mRNA was markedly suppressed by long-term cyclosporin A exposure, whereas accumulation of another lysosomal enzyme N-acetyl-beta-D-glucosaminidase mRNA, which is involved in remodeling of gingival epithelium, was not apparently impaired in cyclosporin A-treated cells. Accumulation of matrix metalloprotease-1 (MMP-1) and tissue inhibitor of matrix metalloprotease-1 (TIMP-1) mRNA, which are involved in remodeling of extracellular matrix, also was not impaired. In addition, we demonstrated that long-term cyclosporin A exposure significantly suppressed not only the activity of the active form of cathepsin-(B + L) compared to the activity in non-treated cells (p = 0.0458), but also the activity of the active form of cathepsin-B (p < 0.0001) in human gingival fibroblasts. CONCLUSION: The decreased ability of protein degradation by not only cathepsin-L but also cathepsin-B is, at least, one of the several factors developing the cyclosporin A-induced gingival overgrowth.

High glucose enhances interleukin-6-induced vascular endothelial growth factor 165 expression via activation of gp130-mediated p44/42 MAPK-CCAAT/enhancer binding protein signaling in gingival fibroblasts.

Diabetic patients are susceptible to severe inflammatory periodontitis manifesting as swollen gingiva with bleeding, but the underlying mechanism is not well understood. Our purpose was to determine the effect of a high glucose (HG) condition on the interleukin-6/soluble interleukin-6 receptor (IL-6/sIL-6R)-induced activation of signaling and vascular endothelial growth factor (VEGF) expression in human gingival fibroblasts (HGFs). In this study, HGFs were cultured for at least two passages under a normal glucose (NG; 5.5 mM) condition or high glucose (25 mM) condition. Importantly, the HG condition significantly induced expression of gp130 mRNA in HGFs compared with levels in control cells. Consistent with the expression of its mRNA, the HG condition also increased the expression of gp130 protein, and phosphorylation of the tyrosine residue by gp130 was enhanced significantly by IL-6/sIL-6R stimulation. Furthermore, the HG condition enhanced the IL-6/sIL-6R-induced phosphorylation of p44/42 MAPK and led to subsequent activation of CCAAT/enhancer binding protein in nuclei. In contrast, there was no significant difference in phosphorylation of JNK between the HG and NG condition. Interestingly, HGFs increased IL-6/sIL-6R-induced VEGF165 mRNA expression and VEGF165 secretion under the HG condition compared with levels under the NG condition. In contrast, the induction of VEGF165 secretion was partially inhibited by PD98059 (selective p44/42 MAPK inhibitor) under the HG condition. In addition, the VEGF165 secretion was completely inhibited by the combination of PD98059 and SP600125 (JNK inhibitor). Our findings suggest that the HG condition indirectly increases VEGF expression via activation of gp130-mediated p44/42 MAPK-CCAAT/enhancer binding protein signaling in HGFs. Thus, elevated VEGF secretion in HGFs under the HG condition may play a role in the development of the severe periodontitis observed in diabetic patients.

CYP2C polymorphisms, phenytoin metabolism and gingival overgrowth in epileptic subjects.

Previous studies suggested that the onset of phenytoin-induced gingival overgrowth depended on serum phenytoin concentration. Cytochrome P450 2C (CYP2C) plays an important role in phenytoin metabolism. Recently, single nucleotide polymorphisms in the coding region of CYP 2C influencing phenytoin metabolism were identified. The purpose of the present study was to see if CYP 2C polymorphisms might relate to the onset and severity of phenytoin-induced gingival overgrowth. Twenty-eight epileptic patients taking phenytoin aged 15 to 75 (mean age: 42.2 years old, 20 males and 8 females) and 56 unrelated healthy subjects aged 30 to 48 (mean age: 36.8 years old, 48 males and 8 females) were examined for CYP 2C polymorphisms. All epileptic subjects were examined for the degree of gingival overgrowth, daily phenytoin dose and serum phenytoin concentration. The results indicated about 7% of the subjects including epileptic and healthy subjects examined were positive for CYP 2C9*3. However, the degree of gingival overgrowth did not directly correlate with CYP 2C polymorphisms. Nevertheless, the subjects with severer gingival overgrowth exhibited significantly higher serum phenytoin concentration, indicating that phenytoin metabolism is an important determinant for the severity of the disease. Additionally, CYP 2C9*3 carriers exhibited significantly higher serum drug concentration to drug dose. Therefore, we concluded although the gene analysis is not directly related to diagnose the disease itself, it can be utilized in estimating serum phenytoin concentration from drug dose, which in turn serves to predict the future development and clinical course of the disease.

C-jun N-terminal kinase (JNK) inhibitor, SP600125, blocks interleukin (IL)-6-induced vascular endothelial growth factor (VEGF) production: cyclosporine A partially mimics this inhibitory effect.

SUMMARY: Angiogenesis is a common complication of organ-transplant rejection. One of the primary responsible molecules for enhanced angiogenesis is vascular endothelial growth factor (VEGF). Activated protein (AP)-1 is considered to play a key role in the transcription of VEGF. c-jun N-terminal kinase (JNK), one of the MAP kinase family members, plays a critical role in AP-1 activation. Thus, we tested the effect of a novel JNK inhibitor, SP600125, on VEGF production in fibroblasts. SP600125 significantly suppressed interleukin (IL)-6-induced production of VEGF in cultured fibroblasts. Cyclosporine A (CsA), a known in vitro anti-angiogenic reagent, partially mimicked this suppression. In fact, CsA suppressed IL-6-induced phosphorylation of JNK. The results indicate that although both SP600125 and CsA are anti-angiogenic by inhibiting VEGF production by way of a JNK-dependent pathway, the inhibitory effect was much stronger with the novel inhibitor of JNK than with CsA.

Cathepsin-L, a key molecule in the pathogenesis of drug-induced and I-cell disease-mediated gingival overgrowth: a study with cathepsin-L-deficient mice.

Drug-induced gingival overgrowth, the chronic side effect of calcium antagonists, is frequently seen due to the increase in patients with hypertension, although the etiology of the disease is largely unknown. I-cell disease, which accompanies gingival overgrowth, is characterized by a deficiency in UDP-N-acetyl-glucosamine and is classified as one of the lysosomal storage diseases. Here, we hypothesized that a common mechanism may underlie the etiology of gingival overgrowth seen in patients treated with calcium antagonist and in patients with I-cell disease. A calcium antagonist, nifedipine, specifically suppressed cathepsin-L activity and mRNA expression, but not that of cathepsin-B in cultured gingival fibroblasts. The activity of cathepsin-L was suppressed up to 50% at 24 hours after treatment of the cells with the reagent. The selective suppression of cathepsin-L activity appeared not to be dependent on Ca(2+), since treatment of the cells with thapsigargin suppressed both cathepsin-B and -L activity. Mice deficient in the cathepsin-L gene manifested enlarged gingivae. Histological observation of the gingivae demonstrated typical features of acanthosis, a phenotype very similar to that of experimentally induced gingival overgrowth. Since cathepsin-L deficiency was reported to be associated with thickening of the skin, impaired cathepsin-L activity may play a key role in the establishment of skin and gingival abnormalities seen in I-cell disease. In addition, reduced cathepsin-L activity may play an important role in inducing drug-induced gingival overgrowth.