FOXC1 regulates BMP‐SMAD activity in a context dependent manner during osteogenic development events

The forkhead box transcription factor FOXC1 is necessary for bone morphogenetic protein (BMP)‐mediated induction of early skeletogenic patterning events. We hypothesize that FOXC1 and SMAD proteins interact to regulate BMP signalling necessary for normal skeletal development. First we demonstrate that FOXC1 and SMAD5 physically interact through co‐immunoprecipitation experiments. Next we show that BMP2 enhances activation of a FOXC1‐responsive reporter gene. We tested whether FOXC1 could mediate signalling of a BMP2‐responsive reporter (2xBRE‐luc). We find that on its own, FOXC1 has no affect on activation of 2xBRE‐luc. Surprisingly, when BMP2 is added, FOXC1 inhibits activation of this reporter. Moreover, FOXC1 prevents activation of this BMP‐responsive element when SMAD4 and SMAD5 are co‐transfected along with FOXC1. To test whether FOXC1 impacted BMP2 mediated osteogenic differentation, we created C2C12 cell lines stably expressing FOXC1. We find that cells expressing FOXC1 differentiated into alkaline phosphatase‐positive osteoblasts in the absence BMP2, yet treatment of FOXC1 expressing cells with BMP2 led to no appreciable increase in alkaline phosphatase activity. Finally we show that FOXC1 prevents activation of Runx2 expression by BMP2. Together these data indicate that FOXC1 and SMAD5 proteins interact to regulate BMP‐signalling in a context dependent manner.