Regulation of Expression of 1,25D3 –MARRS/ERp57/PDIA3 in Rat IEC-6 Cells by TGFβ and 1,25(OH)2D3
Benjamin Rohe1, Susan E. Safford2, Ilka Nemere3, and Mary C. Farach-Carson1*
1Department of Biological Sciences, University of Delaware, Newark, DE, 19716; 2 Department of Biology, Lincoln University, Lincoln University, PA; 3Department of Nutrition and Food Sciences, Utah State University, Logan, UT
* Corresponding author
Tel: 302 831 2277
Fax: 302 831 2281
Email: farachca@udel.edu
Running title: 1,25D3 –MARRS Expression in Rat IEC-6 Cells Abstract
We examined the transcriptional regulation of expression of the redox-sensitive Membrane-Associated Rapid Response, Steroid-binding (1,25D3-MARRS) protein specific for 1,25(OH)2D3 in a rat small intestinal cell line, IEC-6, that demonstrates rapid responses to 1,25(OH)2D3. 1,25D3-MARRS binds and is activated by 1,25(OH)2D3, but is not itself up-regulated by treatment with 1,25(OH)2D3, nor is there a vitamin D response element (VDRE) in its proximal promoter. We previously reported that transforming growth factor β (TGFβ) increased steady state levels of 1,25D3-MARRS transcript and protein approximately two-fold (Rohe et al, 2005). To determine if this up-regulation could be attributed to the function of a highly conserved consensus smad 3 binding element present in the proximal promoter of the 1,25D3-MARRS gene, we created a promoter-reporter [SEAP] construct that was responsive to TGFβ (200 pM). Deletion or mutation of the smad 3 element greatly reduced the response of the 1,25D3-MARRS promoter to TGFβ. Subsequent studies found that the smad 3 response element is bound by a protein found in the IEC-6 nuclear extract, most likely smad 2/3. Interestingly, although 1,25(OH)2D3 alone did not increase expression of the 1,25D3-MARRS promoter-reporter, co-treatment of transfected IEC-6 cells with 1,25(OH)2D3 and TGFβ shifted the dose response for the response to a lower concentration (100 pM). We conclude that TGFβ is a transcriptional regulator of 1,25D3-MARRS expression via a functional smad 3 element and that cross-talk with non-classical 1,25(OH)2D3-stimulated pathways occurs. The findings have broad implications for redox sensitive signaling phenomenon including those that regulate phosphate transport in the intestine.
To the uninitiated, this may look like garbage, or hokum, or double-talk. Actually, it is clear, concise (as a good abstract should be), and demonstrates exactly a point I have tried to make about the way science works. The authors of this study had made some initial findings that did not exactly comport with the hypothesis they had initially used to govern their research, and these findings prompted further research using a control variable to determine exactly why their initial hypothesis was incorrect. Below is the sentence in the abstract to which I refer:
To determine if this up-regulation could be attributed to the function of a highly conserved consensus smad 3 binding element present in the proximal promoter of the 1,25D3-MARRS gene, we created a promoter-reporter [SEAP] construct that was responsive to TGFβ (200 pM). Deletion or mutation of the smad 3 element greatly reduced the response of the 1,25D3-MARRS promoter to TGFβ. Subsequent studies found that the smad 3 response element is bound by a protein found in the IEC-6 nuclear extract, most likely smad 2/3. Interestingly, although 1,25(OH)2D3 alone did not increase expression of the 1,25D3-MARRS promoter-reporter, co-treatment of transfected IEC-6 cells with 1,25(OH)2D3 and TGFβ shifted the dose response for the response to a lower concentration (100 pM).
By modifying their research methods to account for the new data, they discovered something they call "interesting" in the metabolism of these rat cells:
Subsequent studies found that the smad 3 response element is bound by a protein found in the IEC-6 nuclear extract, most likely smad 2/3. Interestingly, although 1,25(OH)2D3 alone did not increase expression of the 1,25D3-MARRS promoter-reporter, co-treatment of transfected IEC-6 cells with 1,25(OH)2D3 and TGFβ shifted the dose response for the response to a lower concentration (100 pM). We conclude that TGFβ is a transcriptional regulator of 1,25D3-MARRS expression via a functional smad 3 element and that cross-talk with non-classical 1,25(OH)2D3-stimulated pathways occurs. The findings have broad implications for redox sensitive signaling phenomenon including those that regulate phosphate transport in the intestine.
In other words, the system is more intricate and involved than they initially suspected, and the chemical governors operate in a manner not accounted for in their initial hypothesis. This does not mean that they were "wrong", or that the hypothesis was "false" - they simply had not accounted for all the possible variables and the chemical interactions among the various bits of stuff (I am using non-scientific short-hand here). Their new conclusions, based upon a refinement of their initial hypothesis culled from preliminary research shows that, while the initial hypothesis was fruitful for various research, even with the discordant results that forced further research, these results "have broad implications for redox sensitive signaling phenomenon including those that regulate phosphate transport in the intestine." In other words, their little chemical experiment with rat intestine cells has actually increased the ways in which such research can be used to examine the functioning of chemical processes in digestion.
Is this conclusion 100% accurate? Of course not! Their initial research was certainly not, which is why they went further, introducing a control variable in order to take account of the odd results of their initial findings. One key phrase towards the end of the abstract I want to highlight is the authors describe the mechanism as "most likely" - not definitively, not proven beyond a reasonable doubt, but within the bounds both of their operating hypothesis and the statistical margin of error governing their research, their conclusion is probably right. That their results in this particular research study are fruitful for further research could sharpen that conclusion, or it could overturn it. But, further research being not only possible but likely, changes can occur which the authors themselves call "surprising", as they did of their initial experimental results.
By the way, one of the authors of this study is my sister.
