BERKELEY, CA (UroToday.com) - Calpains (calcium-activated proteases) were initially thought to participate primarily in cellular events in which intracellular calcium levels rise to catastrophically high levels.
In recent years, it has become very clear that physiological increases in calcium, occurring in response to extracellular signals, can activate calpains in a controlled fashion, allowing the protease to participate in the modulation of specific cellular responses. Our paper provides examples of both types of calpain activation, physiologic and non-physiologic, in a human prostate cancer cell line. The results provide new insights into the role of lysophosphatidic acid (LPA) in prostate cancer cells. Several important lessons were learned throughout the course of this study.
| Our report illustrates the importance of the actions of LPA at focal adhesions, specifically in protecting these cells from insults that increase intracellular calcium. |
This paper was a long time in development. The first author, who was a graduate student at the time when the study was initiated, was investigating various effects of LPA on PC-3 cells. LPA species, which are small lipid mediators, are ligands for G protein-coupled receptors. Our lab had already shown that 18:1 LPA acts as a growth factor in prostate cancer cells. We had been studying the regulation of FAK in other types of cell lines, and we therefore performed immunoblots for FAK in PC-3 cells. In these exploratory experiments, we were testing (among other things) which effects of LPA, an agonist that increases intracellular calcium, might be mimicked by ionomycin. Treatment with ionomycin is often used as a "quick and dirty" way to increase intracellular calcium levels in cell culture studies. We used serum-starved cells for the experiments, which is standard practice for examining growth factor action. Removal of serum reduces mitogenic signaling to a "basal" level, enabling investigators to observe the effects of growth factors that are added subsequently. This is particularly important for studies of LPA action, since LPA is one of the major growth factors present in serum. In pilot experiments, we were startled and perplexed to see that ionomycin caused FAK protein to disappear from our immunoblots. Even more surprising was the observation that LPA partially prevented the loss of FAK in ionomycin-treated cells, even though LPA itself could induce slight proteolysis of FAK. Although this unexpected result was not what our experiments had been designed to test, the results were intriguing enough to merit follow-up experiments. Thus, the first lesson is that important observations can arise in a serendipitous fashion.
The simplest interpretation of the ionomycin results was that a calcium-activated protease could degrade FAK. Review of the literature available at the time revealed that FAK could serve as a substrate for calpains, although the significance of this observation had yet to be fully explored. The first author on our study, Dr. Joshua J. Park, completed his MS dissertation by examining the inhibitory effects of LPA, on ionomycin action, in considerable detail. He established that ionomycin only caused substantial degradation of FAK in serum-starved cells, showed that multiple signaling proteins are substrates for calpains, and laid the groundwork for later mechanistic studies. After Dr. Park graduated and moved on to further endeavors, several graduate students and postdoctoral fellows intrepidly took up this study as a "side project," using additional technologies to further define the mechanisms involved. The accumulated data provide evidence that LPA protects against ionomycin-induced cleavage of focal adhesion proteins by stimulating focal adhesion signaling. Fortuitously, our collective understanding of the physiologic roles of calpain and the actions of LPA advanced during the interim, making the story a more cohesive one. The second lesson, then, is that it sometimes takes a long time for an initial observation to be pursued to a satisfying conclusion.
Another insight gleaned from this study concerns the value of "healthy skepticism." In this case, while serum-starved cells remain the gold standard for studying growth factor action, our work demonstrates that these cells are fragile in some respects. In particular, the withdrawal of growth factors makes the focal adhesion complexes very sensitive to calcium-mediated proteolysis, leading to cell detachment. Thus, although serum withdrawal is necessary for studies of growth factor action, it is important to keep in mind that the starved cells are not necessarily in an optimal state prior to re-addition of growth factors. In this particular case, the sensitivity of the serum-starved cells to ionomycin was readily apparent upon light microscopic examination after about 30 minutes of ionomycin treatment, when the cells began to round up and detach from the plate. Therefore, the third lesson is not to take experimental systems for granted, and to always keep an eye on the cells.
Finally, with respect to prostate cancer, several groups, including ours, have gone on to show that LPA plays an important role as a lipid mediator in prostate cancer cells, promoting not only growth but also survival and migration. LPA is produced as an autocrine factor in response to treatment of prostate cancer cells with other growth factors, creating a positive feedback loop that involves bidirectional cross-talk between growth factor receptors. Our report illustrates the importance of the actions of LPA at focal adhesions, specifically in protecting these cells from insults that increase intracellular calcium. The pro-survival actions of LPA, while not unique to prostate cancer cells, nonetheless make LPA receptors potential targets for therapeutic intervention in prostate cancer. The recent development of pharmacologic LPA receptor antagonists has made this a particularly exciting area for continued investigation.
Written by:
Kathryn E. Meier, PhD* as part of Beyond the Abstract on UroToday.com. This initiative offers a method of publishing for the professional urology community. Authors are given an opportunity to expand on the circumstances, limitations etc... of their research by referencing the published abstract.
*Program in Nutrition and Exercise Physiology
Washington State University
Spokane, Washington
More Information about Beyond the Abstract
Our report illustrates the importance of the actions of LPA at focal adhesions, specifically in protecting these cells from insults that increase intracellular calcium.