Govindaraj, K.

Karperien, M.

Post, J. N.

Osteoarthritis and Cartilage

English

University of Twente Research Information

Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534 S173suggests that further optimisation of alginate is required before it couldbe considered a suitable scaffold for the development of cartilage withstructural integrity.Cell Signaling290DANCING TRANSCRIPTION FACTORS: WHAT MAKES SOX9 MOVE?K. Govindaraj, M. Karperien, J.N. Post. Developmental BioEngineering,MIRA Institute for Biomedical Technology & Technical Medicine; Univ. ofTwente, Enschede, NetherlandsPurpose: Osteoarthritis (OA) is a multifactorial, degenerative jointdisease, affecting millions of people worldwide, and for which no cureis available. One of the main obstacles in finding a cure is that thepathophysiology of OA at the molecular level is as yet unknown.Multiple signaling pathways, such as WNT, BMP, TGF-b, FGF, IGF, andHIF are involved and their interplay at various stages of chondrocytedevelopment and disease still needs to be explored. Methods likeqPCR, immunofluorescence and western blotting are traditionallyused to understand signaling mechanisms. Though these methods areversatile, they are indirect and not suitable for live cell analysis. Assuch there is a need for a quick and direct read out of the signalinginterplay. We hypothesize that the mobility of transcription factors isdirectly linked to their activity and that this can be measured by thebiophysical technique Fluorescence Recovery After Photobleaching(FRAP). In principle, the mobility of the transcription factors isdependent on their binding to their targets. For example: An activetranscription factor is transiently bound to DNA, rendering it immo-bile whereas an unbound transcription factor will be more highlymobile.Here, we have applied FRAP to study the mobility of SOX9 (the mastertranscription factor regulating cartilage development and homeostasis)in response to upstream signals, such as those activated by WNT. Inaddition, we perturbed the WNT signaling pathway using the WNTantagonists DKK1 and FRZB that we have previously identified to be keyin controlling the chondrocyte phenotype.Methods: C20/A4 cells, immortalized juvenile coastal chondrocytes,were grown on glass coverslips and transfected with SOX9-mGFP ormGFP as a control. Recombinant proteins, WNT3A, DKK1, and sFRP-3(FRZB), were obtained from R&D systems and were added 24 hourspost-transfection at concentrations between 1 and 200 ng/ml. C20/A4were incubated with the recombinant proteins for at least 30 minutes.FRAP was performed on a Nikon A1 confocal microscope. FRAP meas-urements were performed in at least 15 cells per condition and thevalues were averaged to get the final curve. Results were analyzed usingMatlab™. To correlate protein mobility with DNA binding, Chromatinimmunoprecipitation (ChIP) assays are performed. To study themolecular mechanism underlying increased SOX9 mobility we performprotein half-life determination studies.Results: Treatment of SOX9-mGFP transfected C20/A4 cells withWNT3A significantly decreased the SOX9 immobile fraction from39% to 22% (Fig.1 and Fig.2.). This is in line with observations thatWNT3A treatment reduces the expression of the SOX9 regulatedcartilage specific genes ACAN and COL2A1. Interestingly, the WNTantagonists DKK1 and FRZB did not restore the SOX9 mobility toits control level, but increased the mobility of SOX9 even more. Thisin line with the finding that DKK1 does not restore the geneexpression of ACAN and COL2A1 after WNT treatment of chon-drocytes for 24h.Figure 2. FRAP curve showing change of SOX9-mGFP mobility in responseto WNT3A.Conclusions: We present the application of FRAP for determining thedirect effect of perturbation of signaling pathways on SOX9 mobilityand show that this mobility change correlates to the activity of SOX9. Asa single cell technique, FRAP offers the possibility for investigatingcellular responses to external signals in small samples from for examplebiopsies. We show that FRAP can be used as a quick read-out of SOX9function without the need for long-term experiments. We thereforepropose to use FRAP to help understand the signaling interplay inosteoarthritis pathophysiology and to test potential drug treatments forindividual patients.

Dancing transcription factors: what makes SOX9 move?

K. Govindaraj

M. Karperien

J.N. Post

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Dancing transcription factors: What makes SOX9 move?

https://doi.org/10.1016/j.joca.2016.01.339

Dancing transcription factors: what makes SOX9 move?

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