Structural basis for activation of somatostatin receptor 5 by cyclic neuropeptide agonists.

Somatostatin receptor 5 (SSTR5) is an important G protein-coupled receptor and drug target for neuroendocrine tumors and pituitary disorders. This study presents two high-resolution cryogenicelectron microscope structures of the SSTR5-Gi complexes bound to the cyclic neuropeptide agonists, cortistatin-17 (CST17) and octreotide, with resolutions of 2.7 Å and 2.9 Å, respectively. The structures reveal that binding of these peptides causes rearrangement of a "hydrophobic lock", consisting of residues from transmembrane helices TM3 and TM6. This rearrangement triggers outward movement of TM6, enabling Gαi protein engagement and receptor activation. In addition to hydrophobic interactions, CST17 forms conserved polar contacts similar to somatostatin-14 binding to SSTR2, while further structural and functional analysis shows that extracellular loops differently recognize CST17 and octreotide. These insights elucidate agonist selectivity and activation mechanisms of SSTR5, providing valuable guidance for structure-based drug development targeting this therapeutically relevant receptor.

Immortalization of mouse primary astrocytes.

In cell culture studies, immortalized primary cells have become a useful tool to investigate the molecular and cellular functions of different types of cells. Several immortalization agents, such as human telomerase reverse transcriptase (hTERT) and Simian Virus 40 (SV40) T antigens, are commonly used for primary cell immortalization. Astrocytes, as the most abundant glial cell type in the central nervous system, are promising therapeutical targets for many neuronal disorders, such as Alzheimer's disease and Parkinson's disease. Immortalized primary astrocytes can provide useful information for astrocytes biology, astrocytes-neuron interactions, glial interactions and astrocytes-associated neuronal diseases. In this study, we successfully purified primary astrocytes with immuno-panning method and examined the astrocyte functions after immortalization through both hTERT and SV40 Large-T antigens. As expected, both immortalized astrocytes presented unlimited lifespan and highly expressed multiple astrocyte-specific markers. However, SV40 Large-T antigen, but not hTERT, immortalized astrocytes displayed fast ATP-induced calcium wave in culture. Hence, SV40 Large-T antigen could be a better choice for primary astrocyte immortalization, which closely mimics the cell biology of primary astrocytes in culture. In summary, the purification and immortalization of primary astrocytes presented in this study can be used for studying astrocyte biology under physiological and pathological conditions.

Roles of Stem Cell Exosomes and their MicroRNA Carrier in Bone and Cartilage Regeneration.

Bone and cartilage regeneration is a dynamic and complex process involving multiple cell types, such as osteoblasts, osteoclasts, endothelial cells, etc. Stem cells have been proved to have an efficient capability to promote bone and cartilage regeneration and repair, but the usage of cells harbors some important safety issues, such as immune rejection and carcinogenicity. Exosomes are non-cell structures secreted from various cells. The content of exosomes is enriched with proteins, such as cytoskeleton proteins, adhesion factors, transcription factors, etc., and a variety of nucleic acids, such as mRNA (Messenger RNA), long-chain non-coding RNA, microRNA (miRNA), etc. Exosomes can deliver a variety of contents from the parent cells to the recipient cells in different tissue backgrounds, influencing the phenotype and function of the recipient cells. Recent studies have demonstrated that miRNAs play significant roles in bone formation, suggesting that miRNAs may be novel therapeutic targets for bone and cartilage diseases. Exosomes have been shown with low/no immune rejection in vivo, no carcinogenic risk of infection, nor other side effects. In recent years, stem cell exosomes have been utilized to promote bone and cartilage regeneration processes during bone defect, bone fracture, cartilage repair, osteoporosis, and osteoarthritis. In this review, we discuss different exosomes derived from stem cells and their interactions with target cells, including osteoblasts, chondrocytes and osteoclasts. We also highlight the various signaling pathways involved in stem cell exosome-related bone and cartilage regeneration.

Osteocyte ß3 integrin promotes bone mass accrual and force-induced bone formation in mice.

Background: Cell culture studies demonstrate the importance of ß3 integrin in osteocyte mechanotransduction. However, the in vivo roles of osteocyte ß3 integrin in the regulation of bone homeostasis and mechanotransduction are poorly defined. Materials and methods: To study the in vivo role of osteocyte ß3 integrin in bone, we utilized the 10-kb Dmp1 (dentin matrix acidic phosphoprotein 1)-Cre to delete ß3 integrin expression in osteocyte in mice. Micro-computerized tomography (µCT), bone histomorphometry and in vitro cell culture experiments were performed to determine the effects of osteocyte ß3 integrin loss on bone mass accrual and biomechanical properties. In addition, in vivo tibial loading model was applied to study the possible involvement of osteocyte ß3 integrin in the mediation of bone mechanotransduction. Results: Deletion of ß3 integrin in osteocytes resulted in a low bone mass and impaired biomechanical properties in load-bearing long bones in adult mice. The loss of ß3 integrin led to abnormal cell morphology with reduced number and length of dentritic processes in osteocytes. Furthermore, osteocyte ß3 integrin loss did not impact the osteoclast formation, but significantly reduced the osteoblast-mediated bone formation rate and reduced the osteogenic differentiation of the bone marrow stromal cells in the bone microenvironment. In addition, mechanical loading failed to accelerate the anabolic bone formation in mutant mice. Conclusions: Our studies demonstrate the essential roles of osteocyte ß3 integrin in regulating bone mass and mechanotransduction.

Structural basis for motilin and erythromycin recognition by motilin receptor.

Motilin is an endogenous peptide hormone almost exclusively expressed in the human gastrointestinal (GI) tract. It activates the motilin receptor (MTLR), a class A G protein-coupled receptor (GPCR), and stimulates GI motility. To our knowledge, MTLR is the first GPCR reported to be activated by macrolide antibiotics, such as erythromycin. It has attracted extensive attention as a potential drug target for GI disorders. We report two structures of Gq-coupled human MTLR bound to motilin and erythromycin. Our structures reveal the recognition mechanism of both ligands and explain the specificity of motilin and ghrelin, a related gut peptide hormone, for their respective receptors. These structures also provide the basis for understanding the different recognition modes of erythromycin by MTLR and ribosome. These findings provide a framework for understanding the physiological regulation of MTLR and guiding drug design targeting MTLR for the treatment of GI motility disorders.

Osteocyte ß1 integrin loss causes low bone mass and impairs bone mechanotransduction in mice.

Background: The key focal adhesion protein ß1 integrin plays an essential role in early skeletal development. However, roles of ß1 integrin expression in osteocytes during the regulation of bone homeostasis and mechanotransduction are incompletely understood. Materials and methods: To study the in vivo function of osteocyte ß1 integrin in bone, we utilized the 10-kb Dmp1 (Dentin matrix acidic phosphoprotein 1)-Cre to generate mice with ß1 integrin deletion in this cell type. Micro-computerized tomography, bone histomorphometry and immunohistochemistry were performed to determine the effects of osteocyte ß1 integrin loss on bone mass accrual and biomechanical properties. In vivo tibial loading model was applied to study the possible involvement of osteocyte ß1 integrin in bone mechanotransduction. Results: Loss of ß1 integrin expression in osteocytes resulted in a severe low bone mass and impaired biomechanical properties in load-bearing long bones and spines, but not in non-weight-bearing calvariae, in mice. The loss of ß1 integrin led to enlarged size of lacunar-canalicular system, abnormal cell morphology, and disorientated nuclei in osteocytes. Furthermore, ß1 integrin loss caused shortening and disorientated collagen I fibers in long bones. Osteocyte ß1 integrin loss did not impact the osteoclast activities, but significantly reduced the osteoblast bone formation rate and, in the meantime, enhanced the adipogenic differentiation of the bone marrow stromal cells in the bone microenvironment. In addition, tibial loading failed to accelerate the anabolic bone formation and improve collagen I fiber integrity in mutant mice. Conclusions: Our studies demonstrate an essential role of osteocyte ß1 integrin in regulating bone homeostasis and mechanotransduction. The transnational potential of this article : This study reveals the regulatory roles of osteocyte ß1 integrin in vivo for the maintenance of bone mass accrual, biomechanical properties, extracellular matrix integrity as well as bone mechanobiology, which defines ß1 integrin a potential therapeutic target for skeletal diseases, such as osteoporosis.

TREM2 Regulates High Glucose-Induced Microglial Inflammation via the NLRP3 Signaling Pathway.

Background: TREM2 expressed on microglia plays an important role in modulating inflammation in neurodegenerative diseases. It remains unknown whether TREM2 modulates hyperglycemia-induced microglial inflammation. Methods: We investigated the molecular function of TREM2 in high glucose-induced microglial inflammation using western blotting, qPCR, ELISA, pulldown, and co-IP methods. Results: Our data showed that in high glucose-induced BV2 cells, TREM2 was increased, and the proinflammatory cytokine IL-1ß was increased. TREM2 knockout (KO) attenuated the proinflammatory cytokine IL-1ß; conversely, TREM2 overexpression (OE) exacerbated IL-1ß expression. Furthermore, we found that high glucose promoted the interaction of TREM2 with NLRP3. TREM2 KO abolished the interaction of TREM2 with NLRP3, while TREM2 OE enhanced the interaction. Moreover, TREM2 KO reduced high glucose-induced NLRP3 inflammasome activation, and TREM2 OE augmented high glucose-induced NLRP3 inflammasome activation, indicating that high glucose enhances the expression of TREM2, which activates the NLRP3 inflammasome. To further clarify whether the NLRP3 signaling pathway mediates the TREM2-regulated inflammatory response, we blocked the NLRP3 inflammasome by knocking out NLRP3 and treating cells with a caspase1 inhibitor, which decreased the levels of the IL-1ß proinflammatory cytokine but did not affect the high glucose-induced expression of TREM2. Conclusions: TREM2 modulates high glucose-induced microglial inflammation via the NLRP3 signaling pathway.