Comparison of the effects of oxidative and inflammatory stresses on rat chondrocyte senescence.

Osteoarthritis (OA) is an age-related degenerative joint disease that causes progressive cartilage loss. Chondrocyte senescence is a fundamental mechanism that contributes to the imbalance of matrix homeostasis in OA by inducing senescence-associated secretory phenotype (SASP). Although OA chondrocytes are mainly exposed to oxidative and inflammatory stresses, the role of these individual stresses in chondrocyte senescence remains unclear. In this study, we compared the effects of these stresses on the senescence of rat chondrocytes. Rat chondrocytes were treated with H2O2 and a combination of IL-1ß and TNF-α (IL/TNF) to compare their in vitro effect on senescent phenotypes. For in vivo evaluation, H2O2 and IL/TNF were injected into rat knee joints for 4 weeks. The in vitro results showed that H2O2 treatment increased reactive oxygen species, γ-H2AX, and p21 levels, stopped cell proliferation, and decreased glycosaminoglycan (GAG)-producing ability. In contrast, IL/TNF increased the expression of p16 and SASP factors, resulting in increased GAG degradation. Intraarticular injections of H2O2 did not cause any changes in senescent markers; however, IL/TNF injections reduced safranin O staining and increased the proportion of p16- and SASP factor-positive chondrocytes. Our results indicate that oxidative and inflammatory stresses have significantly different effects on the senescence of rat chondrocytes.

Comparison of the yields and properties of dedifferentiated fat cells and mesenchymal stem cells derived from infrapatellar fat pads.

Introduction: Infrapatellar fat pad (IFP)-derived mesenchymal stem cells (MSCs) have high chondrogenic potential and are attractive cell sources for cartilage regeneration. During ceiling culture to acquire the characteristics of MSCs, mature adipocytes from fat tissue are known to undergo dedifferentiation, generating dedifferentiated fat (DFAT) cells. The purpose of the present study was to compare the yields and biological properties of IFP-derived MSCs and IFP-derived DFAT cells. Methods: IFPs were harvested from the knees of 8 osteoarthritis (OA) patients. DFAT cells were obtained using a ceiling culture of adipocytes isolated from the floating top layer of IFP digestion. MSCs were obtained by culturing precipitated stromal vascular fraction cells. We compared the P0 cell yields, surface antigen profile, colony formation ability, and multipotency of DFAT cells and MSCs. Results: The P0 cell yields per flask and the estimated total cell yields from 1 g of IFP were much greater for MSCs than for DFAT cells. Both MSCs and DFAT cells were positive for MSC markers. No obvious difference was observed in colony formation ability. In differentiation assays, DFAT cells produced greater amounts of lipid droplets, calcified tissue, and glycosaminoglycan than MSCs did. Adipogenic and chondrogenic gene expressions were upregulated in DFAT cells. Conclusions: IFP-derived DFAT cells showed higher adipogenic and chondrogenic potentials than IFP-derived MSCs, but they had a poor cell yield.

Short cytoplasmic isoform of IL1R1/CD121a mediates IL1ß induced proliferation of synovium-derived mesenchymal stem/stromal cells through ERK1/2 pathway.

Objectives: IL1ß enhances proliferation of synovial mesenchymal stem/stromal cells (synMSCs) although they don't express its receptor, IL1R1/CD121a, on the cell surface. This study was aimed to elucidate the underlying mechanisms of IL1ß-mediated growth promotion. Methods: Human synMSCs were isolated from the suprapatellar synovial membrane. Cell proliferation was measured by MTT. Flowcytometric analyses were performed for surface antigen expression. Intracellular signaling pathway was analyzed by western blotting, immunocytochemistry and Q-PCR. Results: IL1ß enhanced proliferation through IL1R1/CD121a because IL1 receptor antagonist (IL1Ra) completely inhibited it. Expression analyses indicated that a short isoform of IL1R1/CD121a is expressed in synMSCs. Immunocytochemistry indicated that IL1R1/CD121a was majorly localized to the cytoplasm. Western blotting indicated that IL1ß induced delayed timing of the ERK1/2 phosphorylation and IκBα degradation in synMSCs. Q-PCR analyses for IL1ß-target genes indicated that cyclin D was specifically downregulated by a MAPK/ERK inhibitor, U0126, but not by a NFκB inhibitor, TPCA-1. In contrast, the expression of inflammatory cytokines such as IL1α and IL6 are significantly decreased by TPCA-1 but less effectively decreased by U0126. Conclusion: Our data indicated that the cytoplasmic IL1R1/CD121a transduced IL1ß signal in synMSCs. And the growth-promoting effect of IL1ß can be separated from its inflammatory cytokine-inducing function in synMSCs.

Clearance of senescent cells with ABT-263 improves biological functions of synovial mesenchymal stem cells from osteoarthritis patients.

BACKGROUND: Osteoarthritis (OA) is an age-related joint disease characterized by progressive cartilage loss. Synovial mesenchymal stem cells (MSCs) are anticipated as a cell source for OA treatment; however, synovial MSC preparations isolated from OA patients contain many senescent cells that inhibit cartilage regeneration through their senescence-associated secretory phenotype (SASP) and poor chondrogenic capacity. The aim of this study was to improve the biological function of OA synovial MSCs by removing senescent cells using the senolytic drug ABT-263. METHODS: We pretreated synovial MSCs derived from 5 OA patients with ABT-263 for 24 h and then evaluated senescence-associated beta-galactosidase (SA-ß-gal) activity, B cell lymphoma 2 (BCL-2) activity, apoptosis, surface antigen expression, colony formation ability, and multipotency. RESULTS: The ABT-263 pretreatment significantly decreased the percentage of SA-ß-gal-positive cells and BCL-2 expression and induced early- and late-stage apoptosis. Cleaved caspase-3 was expressed in SA-ß-gal-positive cells. The pretreated MSCs formed greater numbers of colonies with larger diameters. The expression rate of CD34 was decreased in the pretreated cells. Differentiation assays revealed that ABT-263 pretreatment enhanced the adipogenic and chondrogenic capabilities of OA synovial MSCs. In chondrogenesis, the pretreated cells produced greater amounts of glycosaminoglycan and type II collagen and showed lower expression of senescence markers (p16 and p21) and SASP factors (MMP-13 and IL-6) and smaller amounts of type I collagen. CONCLUSION: Pretreatment of synovial MSCs from OA patients with ABT-263 can improve the function of the cells by selectively eliminating senescent cells. These findings indicate that ABT-263 could hold promise for the development of effective cell-based OA therapy.

CD34+THY1+ synovial fibroblast subset in arthritic joints has high osteoblastic and chondrogenic potentials in vitro.

OBJECTIVE: Synovial fibroblasts (SFs) in rheumatoid arthritis (RA) and osteoarthritis (OA) play biphasic roles in joint destruction and regeneration of bone/cartilage as mesenchymal stem cells (MSCs). Although MSCs contribute to joint homeostasis, such function is impaired in arthritic joints. We have identified functionally distinct three SF subsets characterized by the expression of CD34 and THY1 as follows: CD34+THY1+, CD34-THY1-, and CD34-THY1+. The objective of this study was to clarify the differentiation potentials as MSCs in each SF subset since both molecules would be associated with the MSC function. METHODS: SF subsets were isolated from synovial tissues of 70 patients (RA: 18, OA: 52). Expressions of surface markers associated with MSCs (THY1, CD34, CD73, CD271, CD54, CD44, and CD29) were evaluated in fleshly isolated SF subsets by flow cytometry. The differentiation potentials of osteogenesis, chondrogenesis, and adipogenesis were evaluated with histological staining and a quantitative polymerase chain reaction of differentiation marker genes. Small interfering RNA was examined to deplete THY1 in SFs. RESULTS: The expression levels of THY1+, CD73+, and CD271+ were highest and those of CD54+ and CD29+ were lowest in CD34+THY1+ among three subsets. Comparing three subsets, the calcified area, alkaline phosphatase (ALP)-stained area, and cartilage matrix subset were the largest in the CD34+THY1+ subset. Consistently, the expressions of differentiation markers of the osteoblasts (RUNX2, ALPL, and OCN) or chondrocytes (ACAN) were the highest in the CD34+THY1+ subset, indicating that the CD34+THY1+ subset possessed the highest osteogenic and chondrogenic potential among three subsets, while the differentiation potentials to adipocytes were comparable among the subsets regarding lipid droplet formations and the expression of LPL and PPARγ. The knockdown of THY1 in bulk SFs resulted in impaired osteoblast differentiation indicating some functional aspects in this stem-cell marker. CONCLUSION: The CD34+THY1+ SF subset has high osteogenic and chondrogenic potentials. The preferential enhancement of MSC functions in the CD34+THY1+ subset may provide a new treatment strategy for regenerating damaged bone/cartilage in arthritic joints.

Interscan measurement error of knee cartilage thickness and projected cartilage area ratio at 9 regions and 45 subregions by fully automatic three-dimensional MRI analysis.

BACKGROUND: We have developed a fully automatic three-dimensional MRI analysis software program for automatic segmentation of knee cartilage using a deep neural network. The purpose of this study was to use this software to clarify the interscan measurement error of the knee cartilage thickness and projected cartilage area ratio at 9 regions and 45 subregions in the knee. METHODS: Ten healthy volunteers underwent MRI twice in the same day. The software provided cartilage thickness and projected cartilage area ratio (thickness ≥ 1.5 mm) at 9 regions and 45 subregions of the knee without any manual correction. The interscan measurement error was calculated at each region and subregion from the data of nine donors, except for one donor who had body motion during the MRI examination. RESULTS: The interscan measurement error of cartilage thickness was less than 0.10 mm at all 9 regions and at 39 subregions among 45 subregions. The measurement errors ranged from 0.03 to 0.21 mm. The intraclass correlation coefficients (ICC) of cartilage thickness were higher than 0.75 at all 9 regions and 41 subregions. The interscan measurement error of the projected cartilage area ratio ranged from 0.01 to 0.03 for all 9 regions. CONCLUSIONS: This study clarified the interscan measurement error of the knee cartilage thickness and projected cartilage area ratio.

Mesenchymal Stem Cells in Synovial Fluid Increase in Knees with Degenerative Meniscus Injury after Arthroscopic Procedures through the Endogenous Effects of CGRP and HGF.

BACKGROUND: Mesenchymal stem cells (MSCs) in synovial fluid increase after traumatic meniscus injuries. However, MSC kinetics in synovial fluid may differ for knees with degenerative meniscus injuries. Furthermore, the combination of surgical repair and synovial MSC transplantation has been found to improve clinical symptoms in patients with degenerative meniscus injury, and in this treatment, only the operation procedure without MSC transplantation might increase MSCs in synovial fluid; if so, soluble factors in synovial fluid will be involved. The purpose is this study was to examine whether MSCs exist in synovial fluid of knees with degenerative meniscus injury, to investigate whether MSCs in synovial fluid increase after harvest of synovium and meniscus repair, and to explore what soluble factors in synovial fluids affect the number of MSCs in synovial fluid. METHODS: Subjects were 7 patients with degenerative meniscus injury who underwent meniscal repair and synovial MSC transplantation. Synovial fluid (Pre) was aspirated from knees before harvest of synovium and meniscus repair. After 2 weeks, synovial fluid (Post) was aspirated again before transplantation of synovial MSCs. A half volume of the synovial fluid was plated and cultured for 2 weeks to count the colony formation. The other half was used for antibody array analysis, and the correlation coefficients between the signal intensity and colony number were measured in 503 factors. Factors with high correlation coefficients were verified by migration assay. RESULTS: While cell colonies derived from synovial fluid (Pre) were hardly observed, greater numbers of colonies from synovial fluid (Post) were demonstrated. Of the 503 factors, calcitonin gene-related peptide (CGRP) and hepatocyte growth factor (HGF) had high correlation coefficients between colony number and expression level. Both CGRP and HGF promoted migration of synovial fluid MSCs. CONCLUSIONS: MSCs in synovial fluid were hardly seen in knees with degenerated meniscus injury. They significantly increased 2 weeks after harvest of synovium and meniscus repair. Both CGRP and HGF in synovial fluid can possibly induce MSCs from synovium into synovial fluid. Graphical abstract.

Petaloid recombinant peptide enhances in vitro cartilage formation by synovial mesenchymal stem cells.

In vitro chondrogenesis of mesenchymal stem cells (MSCs) mimics in vivo chondrogenesis of MSCs. However, the size of the cartilage pellets that can be attained in vitro is limited by current methods; therefore, some modifications are required to obtain larger pellets. Petaloid pieces of recombinant peptide (petaloid RCP) have the advantage of creating spaces between cells in culture. The RCP used here is based on the alpha-1 sequence of human collagen type I and contains 12 Arg-Gly-Asp motifs. We examined the effect and mechanisms of adding petaloid RCP on the in vitro chondrogenesis of human synovial MSCs by culturing 125k cells with or without 0.125 mg petaloid RCP in chondrogenic medium for 21 days. The cartilage pellets were sequentially analyzed by weight, sulfated glycosaminoglycan content, DNA retention, and histology. Petaloid RCP significantly increased the weight of the cartilage pellets: The petaloid RCP group weighed 7.7 ± 1.2 mg (n = 108), whereas the control group weighed 5.3 ± 1.6 mg. Sulfated glycosaminoglycan and DNA contents were significantly higher in the petaloid RCP group than in the control group. Light and transmission electron microscopy images showed that the petaloid RCP formed the framework of the pellet at day 1, the framework was broken by production of cartilage matrix by the synovial MSCs at day 7, and the cartilage pellet grew larger, with diffuse petaloid RCP remaining, at day 21. Therefore, petaloid RCP formed a framework for the pellet, maintained a higher cell number, and promoted in vitro cartilage formation of synovial MSCs. © 2018 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. J Orthop Res 37:1350-1357, 2019.

Specific markers and properties of synovial mesenchymal stem cells in the surface, stromal, and perivascular regions.

BACKGROUND: Synovial mesenchymal stem cells (MSCs) are an attractive cell source for cartilage and meniscus regeneration. Synovial tissue can be histologically classified into three regions; surface, stromal and perivascular region, but the localization of synovial MSCs has not been fully investigated. We identified markers specific for each region, and compared properties of MSCs derived from each region in the synovium. METHODS: The intensity of immunostaining with 19 antibodies was examined for surface, stromal, and perivascular regions of human synovium from six osteoarthritis patients. Specific markers were identified and synovial cells derived from each region were sorted. Proliferation, surface marker expression, chondrogenesis, calcification and adipogenesis potentials were compared in synovial MSCs derived from the three regions. RESULTS: We selected CD55+ CD271- for synovial cells in the surface region, CD55- CD271- in the stromal region, and CD55- CD271+ in the perivascular region. The ratio of the sorted cells to non-hematopoietic lineage cells was 5% in the surface region, 70% in the stromal region and 15% in the perivascular region. Synovial cells in the perivascular fraction had the greatest proliferation potential. After expansion, surface marker expression profiles and adipogenesis potentials were similar but chondrogenic and calcification potentials were higher in synovial MSCs derived from the perivascular region than in those derived from the surface and stromal regions. CONCLUSIONS: We identified specific markers to isolate synovial cells from the surface, stromal, and perivascular regions of the synovium. Synovial MSCs in the perivascular region had the highest proliferative and chondrogenic potentials among the three regions.

Complete human serum maintains viability and chondrogenic potential of human synovial stem cells: suitable conditions for transplantation.

BACKGROUND: In our clinical practice, we perform transplantations of autologous synovial mesenchymal stem cells (MSCs) for cartilage and meniscus regenerative medicine. One of the most important issues to ensuring clinical efficacy involves the transport of synovial MSCs from the processing facility to the clinic. Complete human serum (100% human serum) is an attractive candidate material in which to suspend synovial MSCs for their preservation during transport. The purpose of this study was to investigate whether complete human serum maintained MSC viability and chondrogenic potential and to examine the optimal temperature conditions for the preservation of human synovial MSCs. METHODS: Human synovium was harvested from the knees of 14 donors with osteoarthritis during total knee arthroplasty. Passage 2 synovial MSCs were suspended at 2 million cells/100 µL in Ringer's solution or complete human serum at 4, 13, and 37 °C for 48 h. These cells were analyzed for live cell rates, cell surface marker expression, metabolic activity, proliferation, and adipogenic, calcification, and chondrogenic differentiation potentials before and after preservation. RESULTS: After preservation, synovial MSCs maintained higher live cell rates in human serum than in Ringer's solution at 4 and 13 °C. Synovial MSCs preserved in human serum at 4 and 13 °C also maintained high ratios of propidium iodide- and annexin V- cells. MSC surface marker expression was not altered in cells preserved at 4 and 13 °C. The metabolic activities of cells preserved in human serum at 4 and 13 °C was maintained, while significantly reduced in other conditions. Replated MSCs retained their proliferation ability when preserved in human serum at 4 and 13 °C. Adipogenesis and calcification potential could be observed in cells preserved in each condition, whereas chondrogenic potential was retained only in cells preserved in human serum at 4 and 13 °C. CONCLUSION: The viability and chondrogenic potential of synovial MSCs were maintained when the cells were suspended in human serum at 4 and 13 °C.

Yields and chondrogenic potential of primary synovial mesenchymal stem cells are comparable between rheumatoid arthritis and osteoarthritis patients.

BACKGROUND: Mesenchymal stem cells derived from the synovial membrane (synovial MSCs) are a candidate cell source for regenerative medicine of cartilage and menisci due to their high chondrogenic ability. Regenerative medicine can be expected for RA patients with the inflammation well-controlled as well as OA patients and transplantation of synovial MSCs would also be a possible therapeutic treatment. Some properties of synovial MSCs vary dependent on the diseases patients have, and whether or not the pathological condition of RA affects the chondrogenesis of synovial MSCs remains controversial. The purpose of this study was to compare the properties of primary synovial MSCs between RA and OA patients. METHODS: Human synovial tissue was harvested during total knee arthroplasty from the knee joints of eight patients with RA and OA respectively. Synovial nucleated cells were cultured for 14 days. Total cell yields, surface markers, and differentiation potentials were analyzed for primary synovial MSCs. RESULTS: Nucleated cell number per 1 mg synovium was 8.4 ± 3.9 thousand in RA and 8.0 ± 0.9 thousand in OA. Total cell number after 14-day culture/1 mg synovium was 0.7 ± 0.4 million in RA and 0.5 ± 0.3 million in OA, showing no significant difference between in RA and OA. Cells after 14-day culture were mostly positive for CD44, CD73, CD90, CD105, negative for CD45 both in RA and OA. There was no significant difference for the cartilage pellet weight and sGAG content per pellet between in RA and OA. Both oil red O-positive colony rate and alizarin red-positive colony rate were similar in RA and OA. CONCLUSIONS: Yields, surface markers and chondrogenic potential of primary synovial MSCs in RA were comparable to those in OA. Synovium derived from RA patients can be the cell source of MSCs for cartilage and meniscus regeneration.

TNFα promotes proliferation of human synovial MSCs while maintaining chondrogenic potential.

Synovial mesenchymal stem cells (MSCs) are a candidate cell source for cartilage and meniscus regeneration. If we can proliferate synovial MSCs more effectively, we can expand clinical applications to patients with large cartilage and meniscus lesions. TNFα is a pleiotropic cytokine that can affect the growth and differentiation of cells in the body. The purpose of this study was to examine the effect of TNFα on proliferation, chondrogenesis, and other properties of human synovial MSCs. Passage 1 human synovial MSCs from 2 donors were cultured with 2.5 x 10-12~10-7 g/ml, 10 fold dilution series of TNFα for 14 days, then the cell number and colony number was counted. The effect of the optimum dose of TNFα on proliferation was also examined in synovial MSCs from 6 donors. Chondrogenic potential of synovial MSCs pretreated with TNFα was evaluated in 6 donors. The expressions of 12 surface antigens were also examined in 3 donors.2.5 ng/ml and higher concentration of TNFα significantly increased cell number/dish and cell number/colony in both donors. The effect of 25 ng/ml TNFα was confirmed in all 6 donors. There was no significant difference in the weight, or amount of glycosaminoglycan and DNA of the cartilage pellets between the MSCs untreated and MSCs pretreated with 25 ng/ml TNFα. TNFα decreased expression rate of CD 105 and 140b in all 3 donors. TNFα promoted proliferation of synovial MSCs with increase of cell number/ colony. Pretreatment with TNFα did not affect chondrogenesis of synovial MSCs. However, TNFα affected some properties of synovial MSCs.

Pretreatment with IL-1ß enhances proliferation and chondrogenic potential of synovium-derived mesenchymal stem cells.

BACKGROUND AIMS: Synovial mesenchymal stem cells (MSCs) are an attractive cell source for cartilage regeneration because of their high proliferative ability and chondrogenic potential. We have performed clinical trials using synovial MSCs to regenerate articular cartilage. To achieve good clinical outcomes for cell transplantation therapy, it is important to control both quantity (cell number) and quality (pluripotency or chondrogenic potential) of the cells for transplantation. Interleukin (IL)-1ß is a pro-inflammatory cytokine with significant pro-proliferative potential for mesenchymal cells. However, the effects of IL-1ß on synovial MSCs remain unknown. We investigated the effects of pretreatment with IL-1ß on synovial MSCs. METHODS: Human synovial tissue was harvested during total knee arthroplasty. Nucleated cells were plated and cultured in the absence or presence of IL-1ß at 10-13, 10-12, 10-11, 10-10, 10-9 or 10-8 g/mL for 14 days. RESULTS: The number of synovial MSCs increased in a concentration-dependent manner. When cultured for 21 days in chondrogenic medium after pretreatment with 10-8 g/mL IL-1ß, pellet aggregation was observed, whereas pretreatment with 10-12, 10-11 or 10-10 g/mL IL-1ß significantly increased the weight of cartilage pellets (P <0.01). Surface markers for adhesion ability and pluripotency were reduced with high concentrations of IL-1ß. IL-6 and IL-8 expression increased, but no changes in the expression level of growth factors were indicated by cytokine array. CONCLUSIONS: We have demonstrated that pretreatment of IL-1ß increased the proliferation and chondrogenic potential of synovial MSCs, which may promote the regenerative potential of synovial MSCs.

Platelet-derived growth factor (PDGF)-AA/AB in human serum are potential indicators of the proliferative capacity of human synovial mesenchymal stem cells.

INTRODUCTION: For expansion of human mesenchymal stem cells (MSCs), autologous human serum is safer than fetal bovine serum in clinical situations. One of the problems with the use of autologous human serum is that its proliferative effect on MSCs varies widely between donors. The threefold goals of this study were: (1) to demonstrate an improved method for preparing human serum; (2) to identify growth factors predictive of proliferative potential; and (3) to identify a cytokine to promote MSC proliferation in human serum. METHODS: Fresh blood was collected using a closed bag system containing glass beads. The bag was shaken at 20 °C for 30 minutes for rapid preparation, or kept stationary at 4 °C for 24 hours for slow preparation. Passage 0 synovial MSCs derived from four donors were cultured with 10 % conventional rapid preparation serum or modified slow preparation serum from four different donors. To perform the colony-forming unit assay, synovial MSCs were cultured in these serums. The protein expression profile in serum was analyzed using cytokine array. The candidate proteins were speculated from the correlation between the colony-forming ability and protein expression. As an evaluation of the candidate proteins, proliferation ability, surface marker phenotype and differentiation capability of synovial MSCs were examined. RESULTS: Compared with rapid preparation serum, slow preparation serum resulted in a significantly higher total colony number and twofold higher expression levels of nine proteins (angiopoietin-1, BDNF, EGF, ENA-78, IGFBP-2, platelet-derived growth factor (PDGF)-AA, PDGF-AB/BB, RANTES and TfR). Colony number was positively correlated with PDGF-AA/AB concentrations. Exogenous PDGF-AA significantly promoted proliferation of synovial MSCs, whereas PDGF receptor (PDGFR) inhibitor decreased it. Addition of PDGFs or PDGFR inhibitor did not affect surface epitopes of synovial MSCs. Pretreatment with PDGFs or PDGFR inhibitor did not affect chondrogenic, adipogenic, or calcification potentials of synovial MSCs. CONCLUSION: Slow preparation serum contained higher concentrations of PDGF-AA/AB and increased the colony formation number of synovial MSCs. PDGF-AA/AB were indicators of the proliferative potential of human serum. Exogenous PDGF-AA increased proliferation of synovial MSCs without alteration of surface epitopes and differentiation potentials.

Distribution of regulator of G protein signaling 8 (RGS8) protein in the cerebellum.

The regulator of G protein signaling (RGS) proteins modulate heterotrimeric G protein signaling. RGS8 was identified as a brain-specific RGS protein of 180 amino acids. Biochemical studies indicated that RGS8 binds to Galphao and Galphai3, and that it functions as a GTPase-activating protein (GAP) for Galpha subunits. Physiological investigations demonstrated that RGS8 is not a simple negative regulator, but accelerates the G-protein-coupled responses. In situ hybridization analysis showed a highly dense expression of RGS8 mRNA in Purkinje cells of the cerebellum in rat brain. When the cellular distribution of RGS8 was examined in non-neural cells transfected with RGS8 cDNA, the protein was found to be concentrated in nuclei. Further, co-expression of constitutively active Galphao resulted in the translocation of RGS8 protein to the plasma membrane. The cellular distribution of the RGS8 protein in cerebellar Pukinje cells was also studied in detail. It was shown that the protein is excluded from the nuclei and distributed in the cell body and dendrites except the axons of Purkinje cells. Thus, it is evident that there is a novel mechanism controlling the distribution of RGS8 protein in cerebellar Purkinje cells.