The identification of cis-regulatory elements and their cognate trans-acting molecules in the proximal promoter which enhance the expression of superficial zone protein

Superficial zone protein (SZP), also known as lubricin, is a major lubricating protein in synovial joints. SZP is expressed by superficial zone chondrocytes, but not by middle or deep zone chondrocytes in articular cartilage. The molecular mechanism behind SZP expression remains unclear and no transcription factors directly acting on the SZP gene promoter have been identified. To identify transcription factors which control the expression of SZP, luciferase reporter assays were used to screen the proximal promoter of the SZP gene, which showed relatively high conservation across species. Then electrophoretic mobility shift assays (EMSAs) were used to confirm the binding of transcription factors to selected regions of the SZP promoter. Transfection of reporter constructs into human articular chondrocytes demonstrated that the promoter fragments spanning the region between -405 to -35 showed highly enhanced luciferase activities, with the region between -142 and -35 having the greatest activity. Small biotinylated DNA probes were synthesized for the region between -142 and -87. The electrophoretic mobility of some DNA probes was retarded by nuclear extracts from human chondrocytes. The probe for the region between -132 and -108 was used for DNA pull down experiments to isolate nuclear proteins which bind to this probe. An LTQ-FT-based LC/MS/MS identified isolated proteins as Ku70 and Ku80, a heterodimeric autoantigen. The binding of Ku70 and 80 to the selected DNA fragment was confirmed through EMSA in the presence of antibodies to these proteins. Endogenous binding of Ku heterodimer to the SZP promoter was also confirmed using chromatin immunoprecipitation. In addition, overexpressed Ku heterodimer increased the expression of SZP whereas the depletion of Ku proteins by their siRNAs led to decreased expression of SZP. In conclusion, the conserved sequences in the promoter region of the PRG4 gene contain transcriptionally active sites and two of the nuclear proteins that bind to this site were Ku70 and Ku80. Ku70 and Ku80 work as a heterodimer and act on an enhancer in the proximal region of SZP promoter.