Last week my former grad student started her post-doc in another time zone. In her honor, I am blogging her first first-author paper which recently came out in PLoS ONE. It's my first open access paper as well!
Irsik DL, Carmines PK, Lane PH (2013) Classical Estrogen Receptors and ERα Splice Variants in the Mouse.
When I started exploring the role of puberty and hormones in the kidney disease of diabetes mellitus (DM), two schools of thought drove the relationship between traditional sex steroids and the kidney:
- Estrogen good
- Testosterone bad
From epidemiological studies, we know that men fare less well than women with many kidney disorders, at least until after menopause. Over time, we have discovered that the world is far more complicated than what we thought. The hormone balance specific for each sex may determine the risk or rate of progression of DM kidney disease. But I am getting ahead of myself.
My hypothesis fell into the "bad testosterone camp." I came upon an opportunity to acquire estrogen receptor alpha knock-out mice, and I conceived an experiment. I would make them diabetic for 2 weeks. Over that time, if estrogen were protective, the knockout mice should get really bad glomerular enlargement, an early marker of DM glomerulopathy. If they did not, then we could feel comfortable rejecting the "estrogen good" hypothesis.
Click to enlarge
This study appeared in 2004 (freely available here) with the surprising finding that the mice lacking estrogen receptors (ER) seemed to be protected from glomerular enlargement. Lack of the full-length ER alpha had no discernible effect in the boys, but the girls had no change in glomerular size with DM, even though their wild-type sisters did (See figure).
This observation led to a lot of reading about estrogen. Two major families of ERs had been identified at that time, designated alpha and beta. Our mouse lacking ER alpha was known to lack hypothalamic feedback, leading to high levels of gonadotropins and circulating estrogen. The existence of ER beta in the kidney was unconfirmed, with many doubting its expression in post-natal life. Undaunted by this supposition, we hypothesized that elevated circulating estrogen, through interactions with ER beta, could lead to the protective effects we demonstrated.
Funding agencies generally agreed that our findings and thoughts were interesting. Not interesting enough to send money, but interesting.
I had almost given up ever finding the answer when Debra Irsik came into my lab, eager to study sex differences in the kidney. We settled on this project, and then I went to a conference given by Zhao-Yi Wang of Creighton University Medical Center. He studied the role of ER alpha splice variants in cell signaling in breast cancer. Splice variants? That do stuff? Did our knock-out have those?
Turns out, they sure could. The initial ER alpha null mouse, created by Lubahn and Korach, did not eliminate these splice variants, further complicating out potential explanations for the initial observation.
We decided our first step would be to find out what ER variants were expressed in normal wild-type mice. After all, Wang's experiments were in breast cancer cell lines; we might not find these in normal cells. Our first paper represents a survey of normal male and female mice for these receptors, with special attention to the kidney.
The full length ER alpha 66 was mainly present in female reproductive tissues but was also found in non-reproductive tissues at lower levels. ER alpha 46 was most highly expressed in the heart of both sexes. ER alpha 36 was highly expressed in the kidneys and liver of female mice but not in the kidneys of males. ER beta was most abundant in non-reproductive tissues and in the ovaries.
Because ER alpha 36 has a unique C-terminus, Wang was able to create an antibody specific for this variant. No antibody for the ER alpha 46 variant can be made. We were able to use an N-terminus antibody to localize ER alpha 66 by immunofluorescent confocal microscopy.
ER alpha 36 localization; Click to enlarge
In female mice, ER alpha 66 showed up in blood vessels, glomeruli, proximal tubular brush border, and the cortical collecting duct. ER alpha 36 localized to the mesangial cells, tubular epithelia, and podocytes (see the pretty immunofluorescent pictures in the figure). ER beta also localized in mesangial cells and podocytes. The boys showed little staining for ER alpha 66; the guys did stain for ER alpha 36 in measangial cells and tubular epithelia. Male mesangial cells also demonstrated ER beta staining.
So what is the bottom line here? First, estrogen receptors are much more complicated that initially thought, with at least two known functional splice variants for the alpha receptor. Makes you wonder how many more of those "junk bands" on western blots may be doing something in vivo! Also, estrogen is far more than a "sex hormone." Sure, its receptors are expressed at far higher levels in female reproductive tissues, but they are still hanging out in other organs, just waiting for that hormone to come around. And those are only the receptors we have identified so far!
Now we have a much more complicated story to tease out; can we possibly isolate the role of splice variants in an in vivo model? Stay tuned for Deb's next paper!