Team Cohen Research Interests

My research interests are as eclectic as the students who are a part of my research group. My “bread and butter” is studying the molecular biology and biochemistry of the human follicle stimulating hormone receptor. However, I have several other passions:

My first true love in science was the steroid hormone cortisol. I had been studying Cushing’s Syndrome since I was 12 because my mother was afflicted with this disease. This had led to 3 distinct research projects in my lab that have been worked on by multiple students:

Since my time at Evident Technologies, I have been fascinated with quantum dots (QD)and what they can do.  These unique semiconductor nanocrystals have some remarkable properties that make them unique in the world of fluorophores- they have a broad Stokes’ shift and very specific emission wavelengths. For more information about my research on quantum dot applications look here.

 

Epigenetic regulation of G protein-coupled receptor expression

Role of ectopic expression of G protein receptors in ACTH-independent adrenal hyperplasia.

ACTH normally signals through the Melanocortin type 2 receptor which is linked to Gs.  Signaling through this receptor increases cellular cAMP levels.  This increase results in production of cortisol in the adrenal cortex.  Did you ever wonder what would happen if some other source caused an increase in cAMP?  Like some other receptor that also is linked to Gs- that receptor would be hijacking the system and telling the cells to make cortisol, even if there was no ACTH present.  It turns out that there several examples of this.  The question is, why?  Why do these receptors start getting expressed in a cell that is not their normal type?  One possibility is that something has changed the silencing mechanisms that usually keep the genes turned off.  We have been investigating the roles of DNA methylation and histone acetylation in regulating the expression of the human LH receptor in adrenal cells.

Background

Cushing’s syndrome is the overproduction of the glucocorticoid hormone cortisol by the adrenal gland.  Normally, adrenocorticotropin (ACTH) produced by the pituitary gland interacts with a G protein coupled receptor (GPCR) on the surface of cells in the adrenal gland to stimulate the production of enzymes required to synthesize cortisol (1).  However, in the case of Cushing’s syndrome, this regulation becomes disrupted.  Sometimes this is the result of overproduction of ACTH by the pituitary gland.  In other cases, the etiology is unclear.  In ACTH-independent Cushing’s syndrome, the adrenal gland overproduces cortisol without ACTH stimulation.

Rationale

In recent years, evidence has grown demonstrating the presence of inappropriately expressed GPCR in the adrenal gland that “hijack” normal signal transduction and allow the adrenal gland to respond to other signals besides ACTH (2).  From the appearance of these aberrant receptors one could infer faulty regulation of gene expression.   I am interested in finding the defect that allows these unrelated receptors to be expressed.

Previous Work

Work from Lacroix et al has shown that it is possible clinically to identify the presence of these extra receptors.  In particular, the human luteinizing hormone receptor has been shown to be correlated with pregnancy induced Cushing’s syndrome (3).  The promoter of the hLHR has been well studied and methylation status of the promoter has been characterized in non-adrenal cells (4).  However, no one has looked at human adrenal cells to determine the methylation status in normal or hypertrophic tissue.  Work done in our lab by Kristen Laramie (Union ’07) has shown that hLHR expression increases in a human adrenal cell line when the cells were treated with inhibitors of cytosine methyltransferase and histone deacetylase. However, much work still needs to be done to establish the cause of demethylation of DNA and acetylation of histones in the pathogenesis of Cushing’s syndrome.

Research Plan

We will continue to utilize the human adrenal cell line we have been working with to determine if hLHR is uniquely regulated by DNA methylation/histone deacetylation, or if more of the ectopic receptors seen in Cushing’s syndrome are also regulated by a similar mechanism.  Treatement with inhibitors of these enzymes will be used to treat the cells and then gene expression will be monitored using reverse transcriptase-PCR to detect changes in mRNA levels.  Interestingly, we have found that the human follicle stimulating hormone receptor is not upregulated when cells are treated with these inhibitors, suggesting that there is some specificity to the derepression.

In addition, I have been working to establish collaborations with labs to obtain tissue samples from human adrenals from patients with Cushing’s syndrome and from a mouse model of LH-dependent Cushing’s syndrome to study the promoter in situ using methylation specific PCR.  This process involves extracting genomic DNA and treating it with sodium bisulfite to deaminate unmethylated cytosine to thymine.  This can be detected using primers specfic to either the cytosine or the thymine to demonstrate if the original base was methylated and therefore protected from the sodium bisulfite.  In this way, the degree and location of methylation in the promoter can be determined and correlated with changes in gene expression.

What We Will Learn

Among the questions we want to address is why patients show variability in their ectopic receptor expression.  If there is a single mechanism leading to derepression, then it would be expected that the defect that leads to inappropriate expression of one GPCR would lead to multiple overexpressed receptors.   However, this is not what is observed clinically.  Since the defects in these patients seem to be limited to the adrenal gland, there must be some common denominator that we can works towards identifying.  Initially, we will work to characterize changes in methylation in tissue derived from multiple types of ACTH independent Cushing’s syndrome, and then look for commonalities.

References

  1. Bourdeau I, Lampron A, Costa MH, Tadjine M, Lacroix A 2007 Adrenocorticotropic hormone-independent Cushing’s syndrome. Curr Opin Endocrinol Diabetes Obes 14:219-225
  2. Christopoulos S, Bourdeau I, Lacroix A 2004 Aberrant expression of hormone receptors in adrenal Cushing’s syndrome. Pituitary 7:225-235
  3. Lacroix A, Hamet P, Boutin JM 1999 Leuprolide acetate therapy in luteinizing hormone–dependent Cushing’s syndrome. N Engl J Med 341:1577-1581
  4. Zhang Y, Fatima N, Dufau ML 2005 Coordinated changes in DNA methylation and histone modifications regulate silencing/derepression of luteinizing hormone receptor gene transcription. Mol Cell Biol 25:7929-7939

Academic stress , cortisol, and depression in college students

This project centers around a few interesting observations: 1. Cortisol is classically known as a “stress” hormone. It is known to increase in response to stress to help your body cope.  2. Depression is linked to cortisol; it is one of the symptoms of Cushing’s Syndrome.  3. Some college students have difficulty dealing with stress and become depressed.  So this got me thinking- in some students, does cortisol correlate with an increased chance of depression?  Obviously you would not expect every student to have identical responses to cortisol, but in some students, perhaps an increase in cortisol with academic stress could represent either a trigger or an early warning of depression.  This may be exacerbated by other factors such as age, gender, diet and exercise, and social stresses.  As we continue the studies, it will be interesting to see how these factors contribute to cortisol and stress.

Cortisol suppression and herbal supplements

Did you hear the one about the herbal supplement that could lower cortisol levels?  There have been lots of ads on TV about pills that could block cortisol and help reduce “belly fat”.  I started to wonder- was there any truth to those claims, and if so, how do they work?  We utilized multiple methods to extract the components of the capsules and then analyzed the extracts by GC/MS.  To our surprise (okay, maybe we weren’t that surprised) there was no clear component that could inhibit cortsiol activity.  In upcoming work, we are looking into an herbal supplement which contains licorice root, a component of which is a known inhibitor of the enzyme 11b-hydroxysteroid dehydrogenase.  Questions abound such as how much of the inhibitor is there?  Is it in a form that can inhibit the enzyme in vitro and in vivo?  We will be working on this in the coming year.

Quantum dot molecular beacons for characterizing gene expression.

Molecular beacons work on the principle of fluorescence resonance energy transfer (FRET).  As fluorophores, energy absorbed by the QDs can be transferred to acceptors like organic fluorophores or quenchers.  We have experimented with each and found that in either approach, energy transfer from the QDs to these acceptors if they are in close proximity (like at the ends of a DNA hairpin).  We have also been able to show that the process can be reversed with a complimentary oligonucleotide.  Next step: demonstrate the utility of the approach in a process mimicking real time PCR.