A dual role for 7-dehydrocholesterol reductase in regulating Hedgehog signalling? (2006)

In a recent paper published in Development, Koide et al. provide evidence for a negative regulatory action of 7-dehydrocholesterol reductase(DCHR7) on the Hedgehog pathway (Koide et al., 2006). In a series of elegant experiments, the authors show that: (1) DHCR7 expression is intimately linked to the expression of Sonic Hedgehog (Shh); (2) DHCR7 functions as a negative regulator of Shh signalling in various model systems; (3) the reductase activity of DHCR7 is indispensable for DCHR7's inhibitory action, whereas the N terminus seems to be essential in this respect; and (4) DHCR7 acts at the level of Smoothened (Smo).These findings surprised us at first, as we recently published that mouse patched 1 (Ptch1) secretes vitamin D3, which subsequently binds to and inhibits Smo (Bijlsma et al.,2006). A consequence of our findings is that a defect in (or the absence of) DHCR7 that leads to the accumulation of its substrate,7-dehydrocholesterol (7-DHC, the precursor of vitamin D3), should inhibit Smo activity (through enhanced Ptch1-dependent vitamin D3 secretion). Indeed, we have shown that the DHCR7 inhibitor AY-9944 or the genetic absence of DHRC7[using Dhcr7-/- cells(Cooper et al., 2003)] results in the increased repression of Smo. By contrast, however, the experiments performed by Koide et al. indicate that knocking down or pharmacologically inhibiting DHCR7 has a stimulatory effect on the Hedgehog pathway rather than an inhibitory one, indicating that DHCR7 acts as a negative regulator of the Hedgehog pathway. Thus, these data apparently conflict with our work, and also with earlier papers that suggest a positive regulatory role for DHCR7 on the Hedgehog pathway. Obviously these paradoxical results raise questions.Upon further critical evaluation of the paper of Koide et al., we could appreciate several subtle differences in experimental set-up that explain most of the apparent discrepancies. For instance, most of the experiments Koide et al. presented were performed in the presence of exogenous Shh. As a result,the effect of Ptch1 on Smo activity was not taken into account and the inhibitory role of vitamin D3 (inhibiting Smo after translocation across the cell membrane) was thus overlooked. In such a model system, changing the levels of intracellular 7-DHC and, consequently, of vitamin D3 by modulating DHCR7 levels does not increase the extracellular vitamin D3 concentration and cannot mediate Smo inhibition. It would be interesting to see the effect of DHCR7 knock down on Hedgehog pathway reporter activity in the absence of Shh.Another complicating factor in the interpretation of the effects of DHCR7 knock down on Hedgehog pathway activity is the ectopic expression of Shh the authors described after DHCR7 morpholino injection [see figures 5 and 6 in Koide et al. (Koide et al.,2006)]. As Shh is not a known target gene of the Hedgehog pathway,this upregulation is puzzling and provides another source of Shh to paralyze Ptch1 in its action as described above. In this regard, it is important to note that in our own experimental set-up, we have aimed to eliminate the contribution of Shh to be able to focus on the action of Ptch1, and once more the differences between models systems in studying Hedgehog signal transduction become apparent.Although (or maybe because) Koide et al. circumvented Ptch1 inhibitory activity in their model system, it was elegantly shown that the reductase activity of DHCR7 is not necessary for the inhibitory action on the Hedgehog pathway. Using various DHCR7 mutant constructs, Koide et al. show that the inhibitory activity of DHCR7 is mediated via the N-terminal domain of DHCR7(Koide et al., 2006). These results should, however, be interpreted with care, as one should realize that the deletion of a large N-terminal part of a protein might remove essential signal- and start-transfer sequences, thereby perturbing the orientation of the protein in the membrane.Combining the data of Koide et al. with our own suggests that DHCR7 plays a dual role in Hedgehog signalling. Through its enzymatic activity, it limits vitamin D3 levels in the cell, thereby reducing vitamin D3 secretion via Ptch1 and, subsequently, Ptch1-dependent Smo inhibition. Alternatively, via a currently unknown mechanism but independently of its enzymatic activity, DHCR7 functions as a negative regulator of Shh signalling. The net effect and physiological relevance of these opposing activities of DHCR7 seems a challenging dilemma that needs to be resolved in the near future. Also, as the authors point out themselves, the duality of DHCR7 action on Hedgehog pathway activity is reflected in the different, often incomprehensible, phenotypes of Smith-Lemli-Opitz syndrome (SLOS) patients.