Initially, four compounds were tested for their effects on MMP and TIMP expression. They were chosen because these compounds were shown to upregulate the expression of MMPs and TIMPs in various tissues and cells, including porcine TM cells and human TM tissues. In our study, these stimuli also upregulated the expression of specific MMPs and TIMPs in human TM cells.
Figure 1 demonstrates the effects of these compounds on the production of proMMPs and TIMPs in TM35D cells. A 24-hour exposure to TPA (25 ng/mL; 40 nM) significantly (
P < 0.05) enhanced the accumulation of proMMP-1 (200% ± 8%, mean ± SEM,
n = 10), proMMP-9 (4918% ± 460,
n = 8), and TIMP-1 (253% ± 31%,
n = 8). ProMMP-1 expression was dependent on, but not linearly correlated with, TPA incubation time; the rate of proMMP-1 accumulation decreased substantially after the first 24 hours
(Fig. 2A) . The stimulatory effect of TPA was concentration-dependent with a calculated EC
50 of 2.62 nM
(Fig. 2B) . The maximal effect was produced by a TPA concentration of 10 nM or higher and ranged from 200% to 500% of concentration in the control. Among the compounds tested, IL-1α (25 ng/mL; 1.4 nM) was the most efficacious in stimulating the expression of proMMP-3 (516% ± 39%,
n = 12; after a 24-hour incubation) in TM35D cells
(Fig. 1) . Its effect was also time- and concentration-dependent with a calculated EC
50 of 0.42 ng/mL (23 pM;
Fig. 3 ). The IL-1α-upregulated MMP-3 expression correlated well with an increase in MMP-3 activity. As shown by zymography
(Fig. 4) , IL-1α produced a concentration-dependent increase in casein hydrolytic activity at a molecular mass of approximately 50 kDa, which is identical with the molecular mass of active MMP-3. IL-1α also stimulated the expression of MMP-9, but did not seem to affect the expression of other MMPs or TIMPs. Similarly, TNFα (25 ng/mL; 1.4 nM) also increased the production of proMMP-3 (300% ± 44%,
n = 12), although its efficacy was lower than that of IL-1α
(Fig. 1) . PDGF-BB (25 ng/mL; 1 nM) upregulated the expression of TIMP-1 only (196% ± 24%,
n = 8;
Fig. 1 ). None of these tested compounds affected the expression of MMP-2 or TIMP-2 in TM35D cells.