Benutzerspezifische Werkzeuge

Progress Report 2013

KFO252_long

Progress Report 2013: Project 3 - Hedgehog signalling in adrenal physiology


Progress towards specific aim 1: Determine the role of Shh in adrenal cell proliferation, hyperplasia and tumour formation

We have successfully developed culture conditions that allow survival and proliferation of adrenal capsule cells, which are likely targets for signaling by Shh from the subcapsular layer.  We have produced these cultures from GliLacZ mice, and used production of LacZ as a readout for Shh signaling. We have shown that Smo agonist increases the expression of both PtcLacZ and Gli1LacZ in these cultures.  We are now in a position to investigate how different Shh isoforms affect pathway activation in these cells, and their effects on proliferation, as well as production of other secreted molecules that may signal to cells below the capsule.  

We have studied the effects Shh signaling on proliferation in the adrenal tumor line NCI-295R.  Thus far, we have shown that neither of the Shh isoforms consistently affects proliferation of these cells.  We are now examining the consequences of perturbing the pathway at more downstream positions using Smo agonists and antagonists. 


Progress towards specific aim 2: Assess the role of Shh in adrenal steroid hormone production

Although the highest levels of Shh signaling in the adrenal appears to be in the capsule (as reflected by Gli1 and Ptc activity), our preliminary data suggested that Shh signaling might have a more direct and acute effect on steroidogenesis itself   We are investigating the effects of Shh signaling on steroidogenesis in both the human adrenal tumor line NCI 295R and whole adrenal cultures from mice.  We began these studies using immunoassays to quantify production of aldosterone, cortisol, corticosterone and DHEA.  Stimulating Shh pathway activity in whole mouse adrenal cultures using Smoothened agonist increases production of corticosterone, but not aldosterone, within 24 hours.  Results from NCI-295R cells are more variable – stimulating Shh signaling increased production of aldosterone and cortisol in some experiments, however other experiments showed no effect.  

In an effort to understand the variability of our results, we are now collaborating with Graeme Eisenhofer’s group to quantify released steroid hormones by mass spectrometry.  While immunoassays may be complicated by cross-reaction with related hormones, mass spectrometry sensitively and specifically detects cortisol, aldosterone and DHEA along with all precursor steroids.   Mass spectrometry is now is providing us with a much richer picture of how steroidogenesis responds to different signals. We find that NCI-295R cells release aldosterone and cortisol, as well as all upstream precursors of these hormones. Stimulating steroidogenesis with forskolin or with angiotensin causes striking dynamic changes in the level of secreted precursor steroids and steroid hormones at different times after stimulation.   These experiments suggest that it is extremely important to distinguish different pathway intermediates from final products, and to study dynamic changes in hormone levels rather than a single time point.  With this in mind, we are now using mass spectrometry to re-examine the effects of Shh on steroidogenesis in both NCI-295R cells and whole adrenal explants.


Progress towards specific aim 3:  Assess the role of Shh in adrenal dysfunction related to metabolic diseases and stress-associated conditions.

We have been collaborating with Trian Chavakis to understand how a high fat diet influences Shh signaling and adrenal function.   In preliminary experiments, we performed QRT-PCR on adrenals from obese and normally fed mice that had been previously generated by the Chavakis lab. These studies showed that expression of both Shh and pathway components are reduced in adrenals of obese mice.  We have now set up new feeding experiments using GliLacZ, PtcLacZ and Shh-GFP reporter mice.  We have established conditions for immunohistochemical and Western detection of Hh pathway components and zone-specific markers in the adrenal.  In the next few weeks we will be able to assess the effects of a high fat diet on adrenal Hh signaling, size and zonation.