mRNA Levels of Membrane-type 1 Matrix Metalloproteinase (MT1-MMP), MMP-2, and MMP-9 and of their Inhibitors TIMP-2 and TIMP-3 in Normal Thyrocytes and Thyroid Carcinoma Cell Lines

Abstract

Thyroid cancer can degrade basement membranes and invade tissues. This depends on a cascade of matrix metalloproteinases involving membrane-type 1 matrix metalloproteinase (MT1-MMP), MMP-2, and MMP-9. We analyzed the expression and role of these MMPs and their specific inhibitors TIMP-2 and TIMP-3 in human highly purified thyroid epithelial, C 643, HTh 74, SW 1736, and 8505 C thyroid carcinoma and thyroid-derived fibroblast cell cultures. The effect of phorbol-myristate acetate (PMA), and of the inflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) on MMP and TIMP mRNA levels were monitored by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) including an internal homologous competitor fragment. The highest MT1-MMP mRNA levels were found in thyroid-derived fibroblasts. The MT1-MMP mRNA expression was increased up to 10-fold by PMA, while all other growth factors tested had only negligible effects. The thyroid carcinoma cells themselves did not seem to play a crucial role in the production of MT1-MMP in thyroid tumors. Higher MMP-2 mRNA levels were found in all cell types investigated. The highest MMP-2 mRNA levels were determined in thyroid-derived fibroblasts and HTh 74 cells. We found a lack of MMP-2 response to IL-1, TNF-alpha, and phorbol esters. In unstimulated cells, MMP-9 mRNA was found near the detection limit or at low levels. In nearly all cell types, treatment with PMA, IL-1, and TNF-alpha caused an increase of the MMP-9 mRNA levels. The results of basal and stimulated MMP-2 and MMP-9 mRNA expression were confirmed at the protein level by gelatin zymography. TIMP-2 and TIMP-3 mRNAs were expressed at high levels. In contrast to the basal TIMP-3 mRNA levels, which varied over a great range, there were no striking differences the cell types from analyzing TIMP-2 mRNA. There were no or only slight stimulatory effects on TIMP-2 and TIMP-3 mRNA expression by IL-1, TNF-alpha, and PMA. Taken together, most enzymes of the MT-MMP/MMP class of proteases facilitating invasion of thyroid tumor cells seem to have been produced by fibroblasts, not by the tumor cells themselves. However, some dedifferentiated thyroid tumor cell lines may be capable of secreting some of these enzymes, as in the case of HTh 74 cells.

Activation of pro-(matrix metalloproteinase-2) (pro-MMP-2) by thrombin is membrane-type-MMP-dependent in human umbilical vein endothelial cells and generates a distinct 63 kDa active species

“…Induced expression occurred over a longer time scale (after 6 h) than the increase in cell-associated MT-MMP activity that was inferred from the presence of the 64 kDa and 62 kDa MMP-2 forms. This is similar to effects of PMA and other angiogenic stimuli on MT1-MMP expression [25,26,35] Another recent study [19] has argued that thrombin activation of pro-MMP-2 in human foreskin microvascular endothelial cells is independent of MT1-MMP. In part, these differences may simply reflect differences in the cell types, since HUVECs spontaneously generate a low level of intermediate and fully active forms of MMP-2 without stimulation.…”

“…Augmentation of the level of the 64 kDa intermediate form by treatment of cells with PMA or con A (both of which increase the expression of MT1-MMP [25,30]), led to a corresponding increase in the 63 kDa species in medium from cells exposed to thrombin together with either con A or PMA as compared with HUVECs treated with thrombin alone. The 64 kDa species was not observed in the conditioned medium of HUVECs stimulated with thrombin, or combinations of thrombin and PMA or con A.…”

Increased expression of CCAAT/enhancer binding protein β correlates with prognosis in glioma patients

“…IL-8, an angiogenic cytokine secreted from glioma cells, is thought to be a causative cytokine of hypervascularity in malignant glioma (23), thus contributing to the glioma progression. On the other hand, IL-6 and IL-8 have been suggested to have a role in cancer cell invasion by the induction of MMPs (matrix metalloproteinase) (32)(33)(34). Degradation of collagen in the extracellular matrix mediated by MMPs has been suggested to play an essential role in cancer invasion (35).…”

Angiogenesis in the thyroid gland

“…Normal thyrocytes in culture express mRNA for MMP-2, whereas thyroid anaplastic carcinoma cell lines express MMP-1, -2 and -9 and TIMP-1, -2 and -3 mRNA in vitro (Aust et al 1997). Interestingly, the thyroid cell type that secretes the greatest amounts of MMPs are fibroblasts, and these are stimulated by phorbol esters to increase secretion of a number of MMPs, including MMP-2 and -9 (Hofmann et al 1998). This would concur with a previous study that found expression of MMP-2 mRNA predominantly restricted to fibroblasts in thyroid tumours (Zedenius et al 1996).…”