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FGFR and Nuclear Receptor Expression in LX-2 Human Hepatic Stellate Cells
Throughout the summer, I worked in the Rutgers Environmental and Occupational Health Sciences Institute (EOHSI) as well as the Rutgers Ernest Mario School of Pharmacy under Dr. Grace Guo. My research was focused on the potential effects of the FGF15 pathway on hepatic stellate cells in hepatic fibrosis.
Hepatic Fibrosis and Hepatic Stellate Cells
Hepatic fibrosis is characterized by excessive scarring within the liver in response to chronic damage. This scarring is caused by the accumulation of connective tissues (collagen), which disrupts hepatic architecture and can potentially lead to cirrhosis.
Within the liver, there are several types of cells such as hepatocytes, Kupffer cells, sinusoidal endothelial cells, etc. However, our study focused on a specific type of cell in the liver: hepatic stellate cells (HSC). Hepatic stellate cells are mesenchymal cells within the space of Disse that transdifferentiate into an activated myo-fibroblast state. As these cells activate, they demonstrate fibrogenic qualities such as increased collagen deposition. They are known as the main extracellular matrix (ECM) producing cells in hepatic fibrosis. Thus, we chose to target these cells for our study.
FGF and FGFR Signaling Pathway
FGF (fibroblast growth factor) and FGFR (fibroblast growth factor receptor) are very crucial proteins in endocrine signaling. We focused on the FGF15-FGFR4 pathway in particular. When bile acids in the ileum bind to farnesoid X receptor (FXR), Fgf15 transcription is induced and travels to the liver. FGF15 will then bind to FGFR4 (or FGFR1) with the assistance of co-receptor β-Klotho. This interaction consequently reduces the expression of cholesterol 7α hydroxylase (Cyp7a1), the gene for the rate-limiting enzyme in bile acid synthesis. It is essentially a feedback inhibition pathway intended to down-regulate bile acids when bile acid content is too high.
Reason for This Study
We focused on this pathway and hepatic stellate cells because our preliminary lab data has shown that FGF15-/- mice (-/- stands for KO) demonstrate decreased levels of liver fibrosis in the event of high-fat diet (HFD) induced nonalcoholic steatohepatitis (NASH). This has lead us to believe that FGF15 may potentially induce liver fibrosis by influencing hepatic stellate cell fibrogenesis through interactions with HSC FGFR. An issue we came across however, was determining whether it is FGF15 that heightens hepatic fibrosis in NASH induced by HFD, or if bile reduces liver fibrosis. As I previously stated, FGF15 up-regulation is responsible for down-regulating bile acids. Thus, it is entirely possible that the increased bile-acid synthesis facilitated through removing the Fgf15 gene is responsible for reducing liver fibrosis, and that FGF15 is an indirect factor. Within this study we did not address this issue, as our main intention was to see if FGF19 (the human ortholog of FGF15) influences hepatic stellate cell fibrogenesis or not. If the FGF19-FGFR ligand complex does facilitate hepatic fibrosis, the goal is to create a FGFR inhibitor that will restrict this interaction.
Cell Culture
We cultured an immortalized LX-2 HSC cell line across 10 passages. Documentation has shown that this particular line of hepatic stellate cells becomes more activated as the cells are passaged. We used trypsin for dissociation and cultured the cells in 2% Fetal bovine serum (FBS) in DMEM (Dulbecco's Modified Eagle Medium). PenStrep and L-glutamine were also added into the culture medium. The cells were generally passaged at 80% confluence, and were counted using a hemocytometer. If you want to learn more about the cell culturing process, you can look at Yesh's blog.
RNA Isolation
After each cell passage, cells from a dedicated flask were resuspended in TRIzol Reagent (crazy dangerous by the way due to its phenol content), which basically destroys cellular membranes (lysis) and makes the RNA accessible. We stored these samples in 1mL tubes at -80 °C until we had time to perform the actual RNA isolation. It is always a good idea to isolate all of your samples at once in order to avoid any inconsistencies.
RNA isolation is extremely simple. The TRIzol-cell suspension was simply spun, and the interface layer (all the RNA) was moved into a separate tube. The RNA solution was then precipitated with isopropyl alcohol, washed a few times with ethanol solutions, and then resuspended in DEPC water (water without RNases). Basically, it's a lot of pipetting and centrifuging: easy, but tedious.
Primer Design
Reverse TranscriptionThe goal of all of this was to analyze gene expression throughout passaging using qPCR (will be explained later). In order to do that however, you need DNA. Thus, we needed to convert the isolated RNA to cDNA (c as in complimentary).
cDNA conversion is a really easy process. You simply create a master mix with your sample RNA, RT enzyme and buffers, dNTPs, DEPC water, and random primer, load them in a thermocycler with a preset temperature cycle for a few hours and there's your cDNA.
In order to carry out a qPCR assay, you also need
specialized primers for the genes you want to track. Previous primers created within the lab for FGFR4 were unspecific (meaning the product created was not just FGFR4). We attempted to design primers using NCBI BLAST. However, we eventually stumbled upon a paper about hepatocellular carcinoma which provided a primer sequence for FGFR4. Our qPCR melt curves were a lot more specific using these primers as you can see in the figure I made.
Real Time Polymerase Chain Reaction (qPCR)
qPCR is probably my favorite assay just because you can use it in just about anything. All you need is your DNA sample, Sybr Green I (which contains buffer, enzyme, etc), and your specific primer. We designed a Viia7 program to analyze gene expression for our specific qPCR plate layout. qPCR uses really small quantities of reagent, but a lot of wells. If you ever want some really intense pipetting practice, load a qPCR plate with a 8 μl reaction volume 364 times (the qPCR machine at Rutgers uses 364 well plates).
The qPCR mechanism is really straightforward. There is an intercalating dye (goes between DNA strands) within the reaction mix. As the DNA is amplified, more and more fluorescence is observed. DNA also doubles each cycle, meaning that CT (cycle threshold) values can be compared to see how gene expression of one protein differs from another. This information is then graphed on the computer program and can be exported to excel. We imported the data into excel, calculated the fold changes for each gene, and graphed them for relative expression.
Our Data and What it Means
Here is an image of our qPCR assay results. As you can see, α-SMA (smooth muscle actin) and COL1α1 both exhibited increases in expression as the cells were passaged. This means that we were successful in activating the cells through serial passaging. Our data for PPARγ was quite unexpected. PPARγ is a nuclear receptor that induces FXR expression. However, PPARg is known to be down- regulated during HSC regulation. Our PPARγ expression data demonstrated a sudden increase in the middle of the passage, which we could not explain. FGFR4 and FGFR1 demonstrated extremely promising results. Both of these receptors demonstrated consistent up-regulation throughout HSC activation (though FGFR4 had a strange cyclic pattern). This means that as HSCs become more activated, more FGF19 receptors are induced and thus increase the potential for FGF19 to influence the cells during hepatic fibrosis.
What's the Next Step?
In the future, we plan on treating cultured hepatic stellate cells with varying dosages of FGF19. We also plan on growing hepatic stellate cells in media recovered from hepatocyte cultures to see how human hepatocyte-produced FGF19 affects HSC fibrogenesis.
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