TSG-6 (
Fig. 2), MMP-1, and MMP-3 (
Fig. 3) were all upregulated by TNF-α and IL-1β in both normal and CCh fibroblasts, suggesting that these three genes were all under the regulation of inflammation. In U373MG cells, the overexpression of C/EBPδ, a transcription factor downstream of NF-κB,
38 also upregulates the transcription of TSG-6, MMP-1, and MMP-3.
39 To resolve the causative relationship between TSG-6 and MMP-1/MMP-3, we used TSG-6 siRNA to downregulate its transcript and protein (
Fig. 4) and disclosed for the first time that one plausible mechanism explaining how MMP-1 is activated in CCh fibroblasts might be the dysregulation of TSG-6. In the absence of IL-1β, TSG-6 siRNA dramatically upregulated the expression of MMP-1 transcript more so than MMP-3 transcript (
Fig. 6) and enhanced the expression of proMMP-1 in cell lysates and actMMP-1 in culture media of normal fibroblasts (
Fig. 6), resulting in an expression pattern resembling that of resting CCh (
Fig. 3). In the presence of IL-1β, these changes were more accentuated. These findings strongly suggest that the expression of TSG-6 is meant to counteract the transcription and activation of MMP-1 particularly. Such an action resembles the anti-inflammatory role of TSG-6 in suppressing MMP-9 activation in rat chemically burned corneas
21 and its chondroprotective role in suppressing MMP activation in the arthritic joint of TSG-6 transgenic mice.
23 In the experimental models of arthritis
17,19 and corneal chemical burns,
21,22 the protective role of TSG-6 is demonstrated by transgenic overexpression and exogenous application of TSG-6 (i.e., through an extracellular route). The likelihood for extracellular TSG-6 to exert its anti-inflammatory action is also suggested by its in vivo constitutive extracellular expression in the normal conjunctival epithelium (
Fig. 1) and the extracellular expression of TSG-6 only by CCh fibroblasts (
Fig. 2). Hence, the pathogenesis of CCh might involve the dysregulation of TSG-6 expression. Further studies are worthwhile to determine how MMP-1 is activated in CCh fibroblasts and whether the activation of MMP-1 in CCh fibroblasts is mediated by the overproduction of reactive oxygen species or serine proteinases known to activate MMP-1
40 and how TSG-6 might control such an activation process.