Tokyo University of Science
Discovery of a new target gene for osteoporosis treatment drug ~ Elucidation of part of the pharmacological action mediated by parathyroid hormone ~
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[Research summary and points]
We have discovered a new gene, Gprc5a, which can serve as a new drug discovery target for osteoporosis treatments.
Gprc5a acts downstream of parathyroid hormone (PTH) signaling and suppresses osteoblast proliferation and differentiation.
It is thought to suppress BMP signal transduction by interacting with bone morphogenetic protein (BMP) type I receptors.
[Image: [Research overview]
A group led by Professor Masayoshi Hayata and Chirei Sanpei (1st year doctoral student) from the Department of Biopharmaceutical Sciences, Faculty of Pharmaceutical Sciences, Tokyo University of Science, has discovered a novel gene, Gprc5a, which could serve as a new drug discovery target for osteoporosis treatments.
Given that the existing osteoporosis drug teriparatide is derived from parathyroid hormone (PTH), Professor Hayata and his colleagues screened for genes that act downstream of PTH signaling and have the potential to be involved in bone formation. We have discovered a new gene, Gprc5a, that encodes a G protein-coupled receptor. Gprc5a has the function of suppressing the proliferation and differentiation of osteoblasts.
Furthermore, Gprc5a gene expression was additively increased by co-treatment of teriparatide and active vitamin D3 or retinoic acid. Furthermore, Gprc5a was suggested to suppress BMP signal transduction by interacting with bone morphogenetic protein (BMP) type I receptors. These results suggest that Gprc5a may function as a negative feedback factor for the bone formation promoting effect of teriparatide. The results of this research were published online in the
international academic journal “Journal of Cellular Physiology” on May 20, 2024.
[Research background]
Osteoporosis is a disease that causes bones to become brittle due to a decrease in bone strength due to a decrease in bone mass and bone quality, leading to fractures. Osteoporosis is a disease commonly seen among the elderly, and it is estimated that 12.8 million people, or 1 in 10 people, suffer from osteoporosis in Japan. Osteoporosis weakens bones and causes fractures, which significantly reduces the quality of life and threatens life expectancy. In Japan, where the population is aging, the prevention and treatment of osteoporosis is an important issue in terms of medical resources and life support for the elderly. Osteoporosis patients need to continue a long-term, accurate drug treatment strategy that is tailored to each individual, including the type, order, and administration period of drugs, and drug selection is especially important. Osteoporosis treatment drugs are divided into two types: bone resorption inhibitors and bone formation promoters. Although effective drugs such as bisphosphonates have been developed to suppress bone resorption, there are only three types of drugs that can promote bone formation: teriparatide, romosozumab, and
abaloparatide. In addition, the mechanism by which these drugs increase bone mass remains unclear, and there is a need to develop more effective therapeutic drugs with new mechanisms of action. Therefore, this research team focused on teriparatide, a peptide derived from parathyroid hormone (PTH) that has a strong bone formation promoting effect, elucidated its pharmacological effects, and searched for new drug targets. PTH originally increases blood calcium concentration by promoting bone resorption via osteoblasts. However, teriparatide is used as an osteogenesis-promoting drug because it exhibits a significant osteogenesis-promoting effect when administered intermittently once a day or once a week.
It is believed that teriparatide promotes osteogenic activity by promoting the differentiation of progenitor cells into osteoblasts and suppressing osteoblast apoptosis. However, the full scope of its pharmacological effects has not yet been elucidated, and there are also patients who do not respond to teriparatide treatment
(non-responders).
What is important in understanding the mechanism of action of teriparatide is that it has both bone-forming and bone-resorbing effects. Bone resorption caused by teriparatide also occurs by inducing osteoclast differentiation signals through the expression of RANKL (*1). Therefore, controlling this signal is necessary to develop more effective treatments. In addition, it is important to further elucidate the molecular pathway by which PTH promotes bone formation. Therefore, in this study, we screened for genes that act downstream of PTH signals and have the potential to be involved in bone formation, and investigated the functions of the discovered genes.
[Details of research results]
・Searching for target genes of parathyroid hormone (PTH) signals Professor Hayata and colleagues treated mouse osteoblast MC3T3-E1 cells with PTH for 1 hour and performed RNA-Seq (*2) to identify early response genes to PTH. When we defined a significant difference as 4-fold or more compared to untreated cells, we identified 175 genes whose expression levels increased and 75 genes whose expression levels decreased due to PTH treatment. Among the genes whose expression level increased, 1. Increase in expression level due to PTH treatment has not been reported so far, 2. It is one of the GPCRs (*3) that is a pharmacological target of many therapeutic drugs, and 3. Osteoblast Professor Hayata and his colleagues focused on Gprc5a for three reasons: its function in cells remains unclear.
・Expression of Gprc5a in the PTH signaling pathway
It is known that when PTH binds to the PTH receptor, several signal transduction pathways such as cAMP (*4), intracellular Ca2 , and protein kinase C are activated. Therefore, we investigated which transduction pathway contributes to Gprc5a expression. As a result, treatment with forskolin, an activator of the cAMP pathway,
significantly increased the expression level of Gprc5a. This suggested that PTH mainly induces Gprc5a expression through the cAMP pathway. ・Additive increase in expression level due to co-treatment of retinoic acid or vitamin D3 and PTH
Gprc5a was first identified as a gene induced by retinoic acid treatment in the human head and neck squamous cell carcinoma cell line UM-SCC-22B. Therefore, we analyzed the effect of retinoic acid on the expression of Gprc5a. Similar experiments were conducted with vitamin D3, which is already used as a treatment for osteoporosis.
The results showed that treatment with retinoic acid or vitamin D3 for 1 hour increased it more than 3 times. More interestingly, Gprc5a expression was additively elevated by co-treatment of PTH and retinoic acid or vitamin D3.
・Suppression of osteoblast proliferation and differentiation by Gprc5a In the MTT test (*5) and BrdU labeling test (*6), knockdown of Gprc5a enhanced the proliferation of MC3T3-E1 cells in the absence of PTH, indicating that Gprc5a suppresses cell proliferation in osteoblasts. It was suggested that.
We also investigated the effect of Gprc5a knockdown on osteoblast differentiation and confirmed that Gprc5a knockdown promoted osteoblast differentiation at early and late stages of
differentiation. These results suggest that Gprc5a suppresses not only osteoblast proliferation but also differentiation.
・Mechanism by which Gprc5a suppresses osteoblast differentiation A database search for proteins that interact with Gprc5a and may be involved in osteoblast differentiation identified BMPR1A, which encodes a type I BMP receptor, also known as activin receptor-like kinase 3 (ALK3). . In fact, it was confirmed that Gprc5a interacts with BMPR1A and that Gprc5a suppresses BMP signaling.
Based on the above results, Gprc5a is expected to be a new target for osteoporosis therapeutics. Professor Hayata, who led this research, said, “As a researcher in biopharmaceutical science, I have long wondered why a hormone that absorbs bone in the body exhibits bone-forming effects when administered externally.” Therefore, our motivation was to focus on osteoblasts and elucidate the
mechanism.This time, we found that although Gprc5a is a gene induced by teriparatide, it has the exact opposite effect of suppressing bone formation. This made me realize that life phenomena have a much more complex mechanism than humans expected.In the future, the results of our research will help improve the quality of life of people suffering from osteoporosis. We hope that this will lead to improved health and longevity.”
*This research was supported by the Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research (JP25462287, JP25670639, JP26253085, JP18K09053, JP21H03381).
【term】
*1 RANKL (Receptor activator of nuclear factor-kappa B ligand) A type of transmembrane protein expressed on the surface of bone cells and osteoblasts. It induces osteoclast differentiation by binding to the transmembrane protein “RANK” expressed on the surface of osteoclast precursor cells.
*2 RNA-Seq (RNA-Sequencing)
A method that comprehensively and quantitatively analyzes gene expression levels in cells and tissues using next-generation sequencers.
*3 GPCR (G protein-coupled receptor)
A general term for membrane proteins that combine with G-proteins within cells, receive stimuli from outside the cell, and trigger cellular responses.
*4 cAMP
A type of cyclic nucleotide and one of the intracellular information transmitters (second messengers).
*5 MTT test
A test that measures the survival rate and proliferation rate of cultured cells using a colorimetric method.
*6 BrdU (Bromodeoxyuridine)
A synthetic nucleotide commonly used to detect proliferative cells in biological tissues.
[Paper information]
Magazine name: Journal of Cellular Physiology
Paper title: Gprc5a is a novel parathyroid hormone-inducible gene and negatively regulates osteoblast proliferation and differentiation Author: Chisato Sampei, Kosuke Kato, Yasuhiro Arasaki, Yuta Kimura, Takuto Konno, Kanon Otsuka, Yukihiro Kohara, Masaki Noda, Yoichi Ezura, Tadayoshi Hayata
DOI: 10.1002/jcp.31297
URL: https://doi.org/10.1002/jcp.31297
*Please note that the system does not allow the use of superscripts, subscripts, special characters, etc., so the notation may differ from the official notation. For the official notation, please refer to the Tokyo University of Science web page
(https://www.tus.ac.jp/today/archive/20240618_2891.html).