Regulation of the Vdr expression by Gli TFs

Previous work of our group indicated that Vdr gene expression is regulated in a Gli3-dependent manner. This was based on the findings that overexpression of Gli3 in wt Ptch fibroblasts led to a significantly increased Vdr expression (Fritsch 2014). Moreover similar results were obtained in Gli2^-/-/Gli3^-/- fibroblasts, which do not show functional expression of any Gli TF, since Gli1 is regulated by Gli2 (Ikram et al. 2004; Regl et al. 2002). Gli3 is usually proteolitically cleaved and mainly acts as a transcriptional repressor and only as a weak activator (Aberger et al. 2012; Roberg-Larsen et al. 2014; Wang et al. 2007b).

However, Gli3 can be shifted towards its full-length activator form Gli3^act by activation of Hh signaling. In order to investigate whether Gli3 can activate Vdr expression we overexpressed Gli3 in wt Ptch or Ptch^-/- cells that show constitutive activation of this pathway (Uhmann et al. 2011a) and wt Ptch cells were additionally incubated with Shh-N-CM. We also used Gli1 -/-/Gli2^-/- fibroblasts in which the effects of Gli3 can be determined without any crosstalk with Gli1 and Gli2. Since the proteolytical processing of Gli3 occurs upstream of the Gli TFs, via phosphorylation of protein kinase A, glycogen synthase kinase 3-beta and casein kinase I (Aberger et al. 2012), we also stimulated the cells with Shh-N-CM to cause a shift towards Gli3^act.

Whereas no significant regulation of Vdr expression was seen in wt Ptch or Ptch -/-cells after Gli3 transfection or Shh-N-CM treatment, the incubation of Gli1^-/-/Gli2^-/- cells with Shh-N-CM resulted in an upregulation of Vdr expression (compare Figs. 5 to 7). Thus, the data suggest that the expression of the Vdr gene is not regulated by Gli3 but may be regulated by Shh in dependency of the cellular context. These results are contradictory to initial experiments by A. Fritsch that showed increased Vdr expression in wt Ptch cells after Gli3 transfection (Fritsch 2014). However, the respective experiments were performed in medium supplemented with 10 % FCS (Fritsch 2014) whereas the experiments presented here were performed with CM that was generated from medium supplemented with 2% FCS. It is known that FCS starvation causes cell cycle arrest (Kronemann et al. 1999) or inhibition of proliferation (Oya et al. 2003). Therefore the differential gene expression pattern might be more likely the result from differential FCS supply than of the presence of Gli3. Moreover, media which are conditioned by cells (e.g. Shh-N-CoM and CM) certainly contain, aside from Shh-N, other factors which are not necessarily present in normal (10% FCS containing)

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media. These factors (e.g. growth factors or soluble ligands) also might affect gene expression levels complicating the comparison between the settings even more.

In Gli1^-/-/Gli2^-/- cells Gli3 is most likely processed to Gli3^rep ((Wang et al. 2007b), reviewed in (Briscoe and Therond 2013)) since Gli1 and Gli2 are completely absent, basal Hh activity is reduced (Lipinski et al. 2008) and Gli3^act is dependent on the Gli1-mediated feedback induction (Bai et al. 2004; Roberg-Larsen et al. 2014). However, Vdr expression was activated after Shh-stimulation in Gli1^-/-/Gli2^-/- cells, which was likewise independent of Gli3 transfection. Whereas Gli3 and its activator form Gli3^act (that should occur after Shh-N-CoM treatment) probably do not play a role in Vdr expression, Shh seems to be able to induce Vdr expression in this cell line. Therefore it is possible that Shh induced a non-canonical pathway, resulting in Vdr gene expression. Such non-canonical effects of Shh have been reported previously for the expression of the Rho GTPases RacI and RhoA, which are regulated in a Shh-dependent, but Gli-independent manner (Polizio et al. 2011). However, because Shh did not enhance Vdr expression in wt Ptch and Ptch^-/- cells, this effect must be specific for Gli1^-/-/Gli2^-/- cells.

Our data furthermore showed that the expression of Gli2 is strongly induced by simultaneous Gli3 overexpression and Shh-N-CM treatment in wt Ptch cells and in Ptch -/-cells after Gli3 transfection. These results suggest that Gli2 expression is induced by Gli3^act. Although Gli3 is widely known as a transcriptional repressor (Marigo and Tabin 1996), others also found that Gli3 can act as an activator of gene expression ( e.g. in the development of the sclerotome (Buttitta et al. 2003), the spinal cord (Bai et al. 2004) or the limbs (Bowers et al.

2012). It is known that Gli3 regulates Gli1 by binding to GliBS in the promoter of Gli1 (Dai et al. 1999), whereas no induction of Gli2 expression by Gli3 has been described yet, probaply because Gli2 does not contain a GliBS in its promoter (Regl et al. 2002). Additionally, we analyzed if the putative GliBS located 312 bp upstream of the first exon of the Vdr gene is functionally active. Although this GliBS is the reverse complement of the consensus sequence (Hallikas et al. 2006; Winklmayr et al. 2010), TF binding to a reverse consencus sequence has been described previously (Scholz et al. 2010). Our analyses of the pmVdrProm^wt reporter constructs showed that overexpression of any Gli TF does not change luciferase activitiy of the pmVdrProm^wt reporter. This supports the findings that the Vdr gene is not directly regulated by Gli TF binding to the Vdr promoter.

Together, these experiments strongly suggest that Vdr expression is not regulated by Gli TFs. Nevertheless, since the Gli code is strongly dependent on the cellular context and

Discussion

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developmental stage (Aberger et al. 2012; Aberger and Ruiz 2014), it is possible that Gli TFs may regulate Vdr expression in other cells lines. Thus, it has been shown that Cyclin D2 is regulated independently of Hh signaling in embryonic kidney development (Hu et al. 2006) but is regulated by Hh signaling during hair follicle development (Mill et al. 2003).

Strikingly, studies of the development of medulloblastoma in mice showed that the mere presence of a GliBS is not sufficient for the expression of the respective gene (Lee et al.

2010). However, whether this also applies for Vdr expression remains to be analyzed in the future. Additionally, a larger proportion of the Vdr promoter and thus more regulatory sequences should be employed in the Vdr promotor plasmids. Since it is an established fact that gene expression is not only regulated by TF binding at the direct vicinity of a promoter (e.g. the Vdr) but also several kb upstream (Hallikas et al. 2006) it is possible that the Vdr gene is rather regulated by such distant enhancers.

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