Arthritis Rheumatol. 2016 Jan;68(1):127-37. doi: 10.1002/art.39337.
Regulation of Cholesterol Homeostasis by Hedgehog Signaling in Osteoarthritic Cartilage.
- 1University of Toronto and Hospital for Sick Children, Toronto, Ontario, Canada.
- 2Hospital for Sick Children, Toronto, Ontario, Canada.
- 3Duke University, Durham, North Carolina.
With no effective therapies to attenuate cartilage degeneration in osteoarthritis (OA), the result is pain and disability. Activation of hedgehog (HH) signaling causes changes related to the progression of OA, with higher levels of Gli-mediated transcriptional activation associated with increased disease severity. To elucidate the mechanism through which this occurs, this study sought to identify genes regulated by HH signaling in human OA chondrocytes.
Using human OA cartilage samples, microarray analyses were performed to detect changes in gene expression when the HH pathway was modulated. Results were analyzed for differentially expressed genes, grouped into functional networks, and validated in independent samples. To investigate the effects of chondrocyte-specific sterol accumulation, we generated mice lacking Insig1 and Insig2, which are major negative regulators of cholesterol homeostasis, under Col2a1 regulatory elements.
HH signaling was found to regulate genes that govern cholesterol homeostasis, and this led to alterations in cholesterol accumulation in chondrocytes. A higher level of Gli-mediated transcription resulted in accumulation of intracellular cholesterol. In genetically modified mice, chondrocyte-specific cholesterol accumulation was associated with an OA phenotype. Reducing cholesterol accumulation attenuated the severity of OA in mice in vivo and decreased the expression of proteases in human OA cartilage in vitro.
HH signaling regulates cholesterol homeostasis in chondrocytes, and intracellular cholesterol accumulation contributes to the severity of OA. Our findings have therapeutic implications, since reduction of HH signaling reversed cholesterol accumulation and statin treatment attenuated cartilage degeneration.
Background: Osteoarthritis is the most common type of arthritis, ranked 11 of 291 conditions that contribute to global disability (1). There is no cure, but research efforts are aimed at elucidating factors that contribute to the development and progression of this disease. Our lab previously identified the Hedgehog signaling pathway to be involved in osteoarthritis (2). Hedgehog signaling normally functions during both prenatal and postnatal life to regulate the development and growth of bones, among other important processes. However, if Hedgehog signaling reaches high levels in the cartilage that lines the bones in our joints, osteoarthritis can result. We wanted to understand why this happens, and hypothesized that Hedgehog signaling was controlling gene expression which ultimately impacted osteoarthritis severity.
Findings: We used cartilage obtained from patients undergoing total knee replacement surgery to identify genes that were regulated by Hedgehog signaling (3). Results from our gene expression studies revealed that several genes involved in cholesterol homeostasis were altered by Hedgehog signaling. We performed experiments using genetically modified mice which confirmed that higher levels of Hedgehog signaling resulted in higher levels of cholesterol biosynthesis in chondrocytes, the major cell type in cartilage. At 6 months of age, these mice displayed characteristic signs of osteoarthritis, including reduced cartilage thickness, changes to underlying bone, increased chondrocyte hypertrophy (swelling), and increased gene expression of enzymes which are known to degrade cartilage. We used two different scoring systems to grade the severity of osteoarthritis and with both, found that genetically modified mice had scores which reflected more severe osteoarthritis in comparison to control littermates. The results from Osteoarthritis Research Society International (OARSI) scoring are shown in Figure 1. This suggested to us that higher levels of cholesterol in the chondrocytes could result in osteoarthritis.
Figure 1. Osteoarthritis Research Society International (OARSI) semi-quantitative scoring for osteoarthritis severity in mouse knee joints (4), where higher scores represent more severe osteoarthritis. Open/white circles represent control mice, and filled/black circles represent genetically modified mice with higher levels of cholesterol in the chondrocytes. 5 mice per group, at 6 months of age. *P<0.05
To determine whether these osteoarthritic changes could be reversed by lowering cholesterol levels in the chondrocytes, we administered lovastatin by implanting a slow-release drug pellet adjacent to the knee in mice. Lovastatin is part of the statin family of drugs which are commonly used to lower cholesterol production (5). We found that after 8 weeks, lovastatin treatment reduced signs of osteoarthritis in mouse knees, regardless of whether osteoarthritis was induced by high levels of Hedgehog signaling, high levels of cholesterol, or surgery. This was evidence to suggest that cholesterol level in the cartilage was mediating the effect of Hedgehog signaling on osteoarthritis severity in mice.
What did this mean for human osteoarthritis? We returned to the human cartilage samples we began with, and looked to see if lovastatin treatment could reduce markers of osteoarthritis. Indeed, we found that administering lovastatin to human cartilage in the laboratory reduced gene expression of enzymes which are known to degrade cartilage. Among these is ADAMTS5, a major enzyme responsible for cartilage degradation in osteoarthritis. We performed additional experiments and found that ADAMTS5 may be directly regulated by mediators of cholesterol homeostasis. This is one potential mechanism through which the level of cholesterol in the chondrocyte may contribute to osteoarthritis severity.
Significance: Although traditionally thought to result from mechanical factors, there is increasing evidence to suggest that chondrocyte homeostasis is disrupted in osteoarthritis. We, and others, have previously demonstrated that Hedgehog signaling is activated in osteoarthritis. In this study we identified cholesterol homeostatic genes as targets of Hedgehog signaling in the cartilage. Higher levels of Hedgehog signaling were associated with accumulation of cholesterol in chondrocytes, and subsequent development of osteoarthritis. Unlike previous studies that reported on systemic cholesterol levels and osteoarthritis, our study focused on what resulted uniquely from chondrocyte-specific cholesterol accumulation and showed a potential mechanism of action in osteoarthritis.
This was the first study to demonstrate that Hedgehog signaling regulates cholesterol homeostatic genes and cholesterol biosynthesis. This is relevant to understanding the function of Hedgehog signaling in controlling cholesterol homeostasis in both normal cartilage biology and disease states. It highlights the role of cellular homeostatic processes in chondrocytes, with practical implications for understanding cartilage pathologies such as osteoarthritis. We demonstrate the efficacy of statin treatment as a pharmacologic that reduces markers of osteoarthritis in cartilage. Because we investigate this using both genetically modified mice and human cartilage, there is the potential for our results to be translated to clinical settings. In fact, statin is currently being investigated for treating human osteoarthritis [Trial Identifier: NCT01645176 (6)].
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