Effects of the selective GPER1 agonist G1 on bone growth

Estrogens may affect bone growth locally or systemically via the known estrogen receptors ESR1, ESR2 and G protein-coupled estrogen receptor 1 (GPER1). Mouse and human growth plate chondrocytes have been demonstrated to express GPER1 and ablation of this receptor increased bone length in mice. Therefore, GPER1 is an attractive target for therapeutic modulation of bone growth, which has never been explored. To investigate the effects of activated GPER1 on the growth plate, we locally exposed mouse metatarsal bones to different concentrations of the selective GPER1 agonist G1 for 14 days ex vivo. The results showed that none of the concentrations of G1 had any direct effect on metatarsal bone growth when compared to control. To evaluate if GPER1 stimulation may systemically modulate bone growth, ovariectomized C57BL/6 mice were treated with G1 or β-estradiol (E2). Similarly, G1 did not influence tibia and femur growth in treated mice. As expected, E2 treatment suppressed bone growth in vivo. We conclude that ligand stimulation of GPER1 does not influence bone growth in mice.

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Nuclear Factor-κB p65 Facilitates Longitudinal Bone Growth by Inducing Growth Plate Chondrocyte Proliferation and Differentiation and by Preventing Apoptosis

Journal of Biological Chemistry ◽

10.1074/jbc.m702991200 ◽

2007 ◽

Vol 282 (46) ◽

pp. 33698-33706 ◽

Cited By ~ 46

Author(s):

Shufang Wu ◽

Janna K. Flint ◽

Geoffrey Rezvani ◽

Francesco De Luca

Keyword(s):

Growth Plate ◽

Bone Growth ◽

Chondrocyte Apoptosis ◽

Pyrrolidine Dithiocarbamate ◽

Growth Plate Chondrocytes ◽

Chondrocyte Proliferation ◽

Longitudinal Bone Growth ◽

Metatarsal Bones ◽

Proliferation And Differentiation ◽

Cultured Chondrocytes

NF-κB is a group of transcription factors involved in cell proliferation, differentiation, and apoptosis. Mice deficient in the NF-κB subunits p50 and p52 have retarded growth, suggesting that NF-κB is involved in bone growth. Yet, it is not clear whether the reduced bone growth of these mice depends on the lack of NF-κB activity in growth plate chondrocytes. Using cultured rat metatarsal bones and isolated growth plate chondrocytes, we studied the effects of two NF-κB inhibitors (pyrrolidine dithiocarbamate (PDTC) or BAY11-7082 (BAY)), p65 short interference RNA (siRNA), and of the overexpression of p65 on chondrocyte proliferation, differentiation, and apoptosis. To further define the underlying mechanisms, we studied the functional interaction between NF-κB p65 and BMP-2 in chondrocytes. PDTC and BAY suppressed metatarsal linear growth. Such growth inhibition resulted from decreased chondrocyte proliferation and differentiation and from increased chondrocyte apoptosis. In cultured chondrocytes, the inhibition of NF-κB p65 activation (by PDTC and BAY) and expression (by p65 siRNA) led to the same findings observed in cultured metatarsal bones. In contrast, overexpression of p65 in cultured chondrocytes induced chondrocyte proliferation and differentiation and prevented apoptosis. Although PDTC, BAY, and p65 siRNA reduced the expression of BMP-2 in cultured growth plate chondrocytes, the overexpression of p65 increased it. The addition of Noggin, a BMP-2 antagonist, neutralized the stimulatory effects of p65 on chondrocyte proliferation and differentiation, as well as its anti-apoptotic effect. In conclusion, our findings indicate that NF-κB p65 expressed in growth plate chondrocytes facilitates growth plate chondrogenesis and longitudinal bone growth by inducing BMP-2 expression and activity.

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Imaging IGF-I uptake in growth plate cartilage using in vivo multiphoton microscopy

Journal of Applied Physiology ◽

10.1152/japplphysiol.00645.2017 ◽

2017 ◽

Vol 123 (5) ◽

pp. 1101-1109 ◽

Cited By ~ 3

Author(s):

Maria A. Serrat ◽

Gabriela Ion

Keyword(s):

Real Time ◽

Growth Plate ◽

Multiphoton Microscopy ◽

Biologically Active ◽

Growth Plate Chondrocytes ◽

Growth Plates ◽

Metatarsal Bones ◽

Wide Range ◽

Igf I

Bones elongate through endochondral ossification in cartilaginous growth plates located at ends of primary long bones. Linear growth ensues from a cascade of biochemical signals initiated by actions of systemic and local regulators on growth plate chondrocytes. Although cellular processes are well defined, there is a fundamental gap in understanding how growth regulators are physically transported from surrounding blood vessels into and through dense, avascular cartilage matrix. Intravital imaging using in vivo multiphoton microscopy is one promising strategy to overcome this barrier by quantitatively tracking molecular delivery to cartilage from the vasculature in real time. We previously used in vivo multiphoton imaging to show that hindlimb heating increases vascular access of large molecules to growth plates using 10-, 40-, and 70-kDa dextran tracers. To comparatively evaluate transport of similarly sized physiological regulators, we developed and validated methods for measuring uptake of biologically active IGF-I into proximal tibial growth plates of live 5-wk-old mice. We demonstrate that fluorescently labeled IGF-I (8.2 kDa) is readily taken up in the growth plate and localizes to chondrocytes. Bioactivity tests performed on cultured metatarsal bones confirmed that the labeled protein is functional, assessed by phosphorylation of its signaling kinase, Akt. This methodology, which can be broadly applied to many different proteins and tissues, is relevant for understanding factors that affect delivery of biologically relevant molecules to the skeleton in real time. Results may lead to the development of drug-targeting strategies to treat a wide range of bone and cartilage pathologies. NEW & NOTEWORTHY This paper describes and validates a novel method for imaging transport of biologically active, fluorescently labeled IGF-I into skeletal growth plates of live mice using multiphoton microscopy. Cellular patterns of fluorescence in the growth plate were completely distinct from our prior publications using biologically inert probes, demonstrating for the first time in vivo localization of IGF-I in chondrocytes and perichondrium. These results form important groundwork for future studies aimed at targeting therapeutics into growth plates.

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The role of the G protein-coupled receptor GPR30 in the effects of estrogen in ovariectomized mice

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.90691.2008 ◽

2009 ◽

Vol 296 (3) ◽

pp. E490-E496 ◽

Cited By ~ 68

Author(s):

S. H. Windahl ◽

N. Andersson ◽

A. S. Chagin ◽

U. E. A. Mårtensson ◽

H. Carlsten ◽

...

Keyword(s):

Growth Plate ◽

Fat Mass ◽

Bone Growth ◽

Distal Femur ◽

Uterine Weight ◽

Marrow Cellularity ◽

G Protein Coupled ◽

Estrogenic Responses

In vitro studies suggest that the membrane G protein-coupled receptor GPR30 is a functional estrogen receptor (ER). The aim of the present study was to determine the possible in vivo role of GPR30 as a functional ER primarily for the regulation of skeletal parameters, including bone mass and longitudinal bone growth, but also for some other well-known estrogen-regulated parameters, including uterine weight, thymus weight, and fat mass. Three-month-old ovariectomized (OVX) GPR30-deficient mice (GPR30−/−) and wild-type (WT) mice were treated with either vehicle or increasing doses of estradiol (E2; 0, 30, 70, 160, or 830 ng·mouse−1·day−1). Body composition [bone mineral density (BMD), fat mass, and lean mass] was analyzed by dual-energy-X ray absorptiometry, while the cortical and trabecular bone compartments were analyzed by peripheral quantitative computerized tomography. Quantitative histological analyses were performed in the distal femur growth plate. Bone marrow cellularity and distribution were analyzed using a fluorescence-activated cell sorter. The estrogenic responses on most of the investigated parameters, including increase in bone mass (total body BMD, spine BMD, trabecular BMD, and cortical bone thickness), increase in uterine weight, thymic atrophy, fat mass reduction, and increase in bone marrow cellularity, were similar for all of the investigated E2 doses in WT and GPR30−/− mice. On the other hand, E2 treatment reduced longitudinal bone growth, reflected by decreased femur length and distal femur growth plate height, in the WT mice but not in the GPR30−/− mice compared with vehicle-treated mice. These in vivo findings demonstrate that GPR30 is not required for normal estrogenic responses on several major well-known estrogen-regulated parameters. In contrast, GPR30 is required for a normal estrogenic response in the growth plate.

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G-Protein-Coupled Estrogen Receptor-1 Positively Regulates the Growth Plate Chondrocyte Proliferation in Female Pubertal Mice

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.710664 ◽

2021 ◽

Vol 9 ◽

Author(s):

Ya-Shuan Chou ◽

Shu-Chun Chuang ◽

Chung-Hwan Chen ◽

Mei-Ling Ho ◽

Je-Ken Chang

Keyword(s):

Estrogen Receptor ◽

G Protein ◽

Growth Plate ◽

Longitudinal Growth ◽

Long Bone ◽

Early Puberty ◽

Growth Plate Chondrocytes ◽

Chondrocyte Proliferation ◽

G Protein Coupled ◽

Estrogen Receptor 1

Estrogen enhances long bone longitudinal growth during early puberty. Growth plate chondrocytes are the main cells that contribute to long bone elongation. The role of G-protein-coupled estrogen receptor-1 (GPER-1) in regulating growth plate chondrocyte function remains unclear. In the present study, we generated chondrocyte-specific GPER-1 knockout (CKO) mice to investigate the effect of GPER-1 in growth plate chondrocytes. In control mice, GPER-1 was highly expressed in the growth plates of 4- and 8-week-old mice, with a gradual decline through 12 to 16 weeks. In CKO mice, the GPER-1 expression in growth plate chondrocytes was significantly lower than that in the control mice (80% decrease). The CKO mice also showed a decrease in body length (crown–rump length), body weight, and the length of tibias and femurs at 8 weeks. More importantly, the cell number and thickness of the proliferative zone of the growth plate, as well as the thickness of primary spongiosa and length of metaphysis plus diaphysis in tibias of CKO mice, were significantly decreased compared with those of the control mice. Furthermore, there was also a considerable reduction in the number of proliferating cell nuclear antigens and Ki67-stained proliferating chondrocytes in the tibia growth plate in the CKO mice. The chondrocyte proliferation mediated by GPER-1 was further demonstrated via treatment with a GPER-1 antagonist in cultured epiphyseal cartilage. This study demonstrates that GPER-1 positively regulates chondrocyte proliferation at the growth plate during early puberty and contributes to the longitudinal growth of long bones.

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The phosphatase inhibitor LB-100 acts synergistically with the NPR2 agonist BMN-111 to improve bone growth

10.1101/2020.09.10.288589 ◽

2020 ◽

Author(s):

Leia C. Shuhaibar ◽

Nabil Kaci ◽

Jeremy R. Egbert ◽

Léa Loisay ◽

Giulia Vigone ◽

...

Keyword(s):

Growth Plate ◽

Bone Growth ◽

Ex Vivo ◽

Growth Factor Receptor ◽

Growth Plate Chondrocytes ◽

Newborn Mice ◽

Cgmp Production ◽

Phosphatase Inhibitor ◽

Proliferative Growth ◽

Severe Short Stature

ABSTRACTActivating mutations in fibroblast growth factor receptor 3 (FGFR3) and inactivating mutations in the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase both result in decreased production of cyclic GMP (cGMP) in chondrocytes and severe short stature, causing achondroplasia (ACH) and acrosomelic dysplasia type Maroteaux, respectively. Previously we showed that an NPR2 agonist BMN-111 (vosoritide) increases bone growth in mice mimicking ACH (Fgfr3Y367C/+), and that in control growth plate chondrocytes, FGFR3 signaling decreases NPR2 activity by dephosphorylating the NPR2 protein. Here we tested whether a phosphatase inhibitor (LB-100) could enhance bone growth in ACH. In ex vivo imaging experiments using a FRET sensor to measure cGMP production in chondrocytes of living tibias from newborn mice, LB-100 counteracts the FGF-induced dephosphorylation and inactivation of NPR2. In ex vivo experiments with Fgfr3Y367C/+ mice, LB-100 in combination with BMN-111 increases the rate of femur growth by ∼25% vs BMN-111 alone, restores chondrocyte terminal differentiation, increases the proliferative growth plate area of the femur, and reduces the activity of the MAP kinase pathway. Our results provide a proof of concept that a phosphatase inhibitor could be used together with an NPR2 agonist to enhance cGMP production as a therapy for ACH.GRAPHICAL ABSTRACT

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Growth plate senescence is associated with loss of DNA methylation

Journal of Endocrinology ◽

10.1677/joe.1.06016 ◽

2005 ◽

Vol 186 (1) ◽

pp. 241-249 ◽

Cited By ~ 25

Author(s):

Ola Nilsson ◽

Robert D Mitchum ◽

Lenneke Schrier ◽

Sandra P Ferns ◽

Kevin M Barnes ◽

...

Keyword(s):

Dna Methylation ◽

Growth Plate ◽

Bone Growth ◽

Indirect Evidence ◽

Adult Size ◽

Growth Plate Chondrocytes ◽

Longitudinal Bone Growth ◽

Proliferative Zone

The overall body size of vertebrates is primarily determined by longitudinal bone growth at the growth plate. With age, the growth plate undergoes programmed senescence, causing longitudinal bone growth to slow and eventually cease. Indirect evidence suggests that growth plate senescence occurs because stem-like cells in the growth plate resting zone have a finite proliferative capacity that is gradually exhausted. Similar limits on replication have been observed when many types of animal cells are placed in cell culture, an effect known as the Hayflick phenomenon. However, we found that the number of population doublings of rabbit resting zone chondrocytes in culture did not depend on the age of the animal from which the cells were harvested, suggesting that the mechanisms limiting replicative capacity of growth plate chondrocytes in vivo are distinct from those in vitro. We also observed that the level of DNA methylation in resting zone chondrocytes decreased with age in vivo. This loss of methylation appeared to occur specifically with the slow proliferation of resting zone chondrocytes in vivo and was not observed with the rapid proliferation of proliferative zone chondrocytes in vivo (i.e. the level of DNA methylation did not change from the resting zone to the hypertrophic zone), with proliferation of chondrocytes in vitro, or with growth of the liver in vivo. Thus, the overall level of DNA methylation decreases during growth plate senescence. This finding is consistent with the hypothesis that the mechanism limiting replication of growth plate chondrocytes in vivo involves loss of DNA methylation and, thus, loss of DNA methylation might be a fundamental biological mechanism that limits longitudinal bone growth in mammals, thereby determining the overall adult size of the organism.

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Indirubin-3′-oxime stimulates chondrocyte maturation and longitudinal bone growth via activation of the Wnt/β-catenin pathway

Experimental & Molecular Medicine ◽

10.1038/s12276-019-0306-3 ◽

2019 ◽

Vol 51 (9) ◽

pp. 1-10

Author(s):

Sehee Choi ◽

Pu-Hyeon Cha ◽

Hyun-Yi Kim ◽

Kang-Yell Choi

Keyword(s):

Growth Plate ◽

Bone Growth ◽

Ex Vivo ◽

Hormone Treatment ◽

Current Approach ◽

Plate Height ◽

Growth Plate Chondrocytes ◽

Longitudinal Bone Growth ◽

Chondrocyte Maturation

Abstract Researchers have shown increased interest in determining what stimulates height. Currently, many children undergo precocious puberty, resulting in short stature due to premature closure of the growth plate. However, the current approach for height enhancement is limited to growth hormone treatment, which often results in side effects and clinical failure and is costly. Although recent studies have indicated the importance of paracrine signals in the growth plate for longitudinal bone growth, height-stimulating agents targeting the signaling pathways involved in growth plate maturation remain unavailable in the clinic. The Wnt/β-catenin pathway plays a major role in the maturation of growth plate chondrocytes. In this study, by using an ex vivo tibial culture system, we identified indirubin-3′-oxime (I3O) as a compound capable of enhancing longitudinal bone growth. I3O promoted chondrocyte proliferation and differentiation via activation of the Wnt/β-catenin pathway in vitro. Intraperitoneal injection of I3O in adolescent mice increased growth plate height along with incremental chondrocyte maturation. I3O promoted tibial growth without significant adverse effects on bone thickness and articular cartilage. Therefore, I3O could be a potential therapeutic agent for increasing height in children with growth retardation.

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Investigation of RARg Signaling in Human Growth-Plate Chondrocytes

10.1542/peds.147.3_meetingabstract.790 ◽

2021 ◽

Author(s):

Joshua M. Abzug ◽

Hongying Tian ◽

Masatake Matsuoka ◽

Danielle A. Hogarth ◽

Casey M. Codd ◽

...

Keyword(s):

Growth Plate ◽

Human Growth ◽

Growth Plate Chondrocytes

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Effect of Pulsed Electromagnetic Field Stimulation on the Growth Plate of the Tibia Bone of Rats: An In Vivo Study

Applied Sciences ◽

10.3390/app11167571 ◽

2021 ◽

Vol 11 (16) ◽

pp. 7571

Author(s):

Yoon-Young Sung ◽

Jae-Woo Shin ◽

Won-Kyung Yang ◽

Min-Jin Kim ◽

Ja-Ik Koo ◽

...

Keyword(s):

Electromagnetic Field ◽

Growth Plate ◽

Bone Growth ◽

Articular Chondrocytes ◽

Growth Promotion ◽

Control Group ◽

Pulsed Electromagnetic Field ◽

Tibia Bone ◽

Pulsed Electromagnetic

Currently, many children undergo precocious puberty, resulting in short stature due to premature closure of the growth plate. Pulsed electromagnetic field (PEMF) stimulation induces cell proliferation of articular chondrocytes. We developed a method for growth promotion using equipment with PEMF. In this study, we aimed to evaluate the effects of PEMF on the growth rate of growth plates using an animal model. An experimental study was conducted on 16 3-week-old rats to validate the effects of the growth care device on growth and development by PEMF stimulation at 28 Hz and 20 Gauss. The tibia bones of the groups with and without PEMF administration were dissected after 10 days, and then, the length of the growth plate of the knee and levels of insulin-like growth factor (IGF)-1 hormone in serum were measured. The length of the growth plate on the tibia bone and the levels of circulating IGF-1 were significantly increased by 25.6% and 13.6%, respectively, in the experimental group to which PEMF was applied compared to those of the control group, without any side effects. These results suggest that PEMF can safely stimulate growth of the growth plate in a non-invasive manner to promote bone growth.

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Catch-Up Growth after Hypothyroidism Is Caused by Delayed Growth Plate Senescence

Endocrinology ◽

10.1210/en.2007-0993 ◽

2008 ◽

Vol 149 (4) ◽

pp. 1820-1828 ◽

Cited By ~ 32

Author(s):

Rose Marino ◽

Anita Hegde ◽

Kevin M. Barnes ◽

Lenneke Schrier ◽

Joyce A. Emons ◽

...

Keyword(s):

Growth Rate ◽

Growth Inhibition ◽

Growth Plate ◽

Bone Growth ◽

Structural Characteristics ◽

Linear Growth Rate ◽

Growth Plate Chondrocytes ◽

Growth Plates ◽

Catch Up ◽

Growth Inhibiting

Catch-up growth is defined as a linear growth rate greater than expected for age after a period of growth inhibition. We hypothesized that catch-up growth occurs because growth-inhibiting conditions conserve the limited proliferative capacity of growth plate chondrocytes, thus slowing the normal process of growth plate senescence. When the growth-inhibiting condition resolves, the growth plates are less senescent and therefore grow more rapidly than normal for age. To test this hypothesis, we administered propylthiouracil to newborn rats for 8 wk to induce hypothyroidism and then stopped the propylthiouracil to allow catch-up growth. In untreated controls, the growth plates underwent progressive, senescent changes in multiple functional and structural characteristics. We also identified genes that showed large changes in mRNA expression in growth plate and used these changes as molecular markers of senescence. In treated animals, after stopping propylthiouracil, these functional, structural, and molecular senescent changes were delayed, compared with controls. This delayed senescence included a delayed decline in longitudinal growth rate, resulting in catch-up growth. The findings demonstrate that growth inhibition due to hypothyroidism slows the developmental program of growth plate senescence, including the normal decline in the rate of longitudinal bone growth, thus accounting for catch-up growth.

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