Sex-specific effects of 17β-estradiol and Dihydrotestosterone (DHT) on growth plate chondrocytes are dependent on both ERα and ERβ and require Palmitoylation to translocate the receptors to the plasma membrane

Abstract Estrogens cause growth plate closure in both males and females, by decreasing proliferation and inducing apoptosis of postproliferative growth plate chondrocytes. In vitro studies using 17β-estradiol (E2) conjugated to bovine serum albumin (E2-BSA) show that rat costochondral growth plate resting zone chondrocytes also respond to E2. Moreover, they are regulated by E2-BSA via a protein kinase C and ERK MAPK signaling pathway that is functional only in female cells. To better understand how E2 regulates apoptosis of growth plate chondrocytes, rat resting zone chondrocytes cells were treated with E2 or E2-BSA. E2 caused apoptosis in male and female resting zone and growth zone chondrocytes in a dose-dependent manner, based on elevated DNA fragmentation, terminal deoxynucleotidyl transferase dUTP nick end labeling staining and caspase-3 activation. E2 also up-regulated p53 and Bax protein (Bcl-2-associated X protein) levels and induced release of cytochrome C from the mitochondria, indicating a mitochondrial apoptotic pathway. The apoptotic effect of E2 did not involve elevated nitric oxide production or MAPKs. It was reduced by ICI 182780, which is an estrogen receptor (ER) antagonist and blocked by antibodies to Erα36, a membrane-associated ER. E2-BSA reduced cell viability and increased caspase-3 activity; ICI 182780 had no effect, but anti-ERα36 antibodies blocked the effect. The results indicate that estrogen is able to directly affect the cell population kinetics of growth plate chondrocytes by regulating apoptosis, as well as proliferation and differentiation in both resting zone and growth zone cells. They also have provided further information about the physiological functions of estrogen on longitudinal bone growth.

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Isolation of a plasma membrane-enriched fraction from collagenase-suspended rachitic rat growth plate chondrocytes

Journal of Orthopaedic Research® ◽

10.1002/jor.1100010312 ◽

1983 ◽

Vol 1 (3) ◽

pp. 319-324 ◽

Cited By ~ 5

Author(s):

William W. Bohn ◽

Ross M. Stein ◽

Howard H. T. Hsu ◽

David C. Morris ◽

H. Clarke Anderson

Keyword(s):

Plasma Membrane ◽

Growth Plate ◽

Growth Plate Chondrocytes ◽

Rat Growth

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Growth-plate chondrocytes respond to 17β-estradiol with sex-specific increases in IP3 and intracellular calcium ion signalling via a capacitative entry mechanism

Steroids ◽

10.1016/j.steroids.2005.04.007 ◽

2005 ◽

Vol 70 (11) ◽

pp. 775-786 ◽

Cited By ~ 8

Author(s):

J EKSTEIN ◽

E NASATZKY ◽

B BOYAN ◽

A ORNOY ◽

Z SCHWARTZ

Keyword(s):

Intracellular Calcium ◽

Growth Plate ◽

Calcium Ion ◽

Growth Plate Chondrocytes ◽

Intracellular Calcium Ion ◽

17Β Estradiol ◽

Entry Mechanism

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Rho-ROCK signaling differentially regulates chondrocyte spreading on fibronectin and bone sialoprotein

AJP Cell Physiology ◽

10.1152/ajpcell.00548.2007 ◽

2008 ◽

Vol 295 (1) ◽

pp. C38-C49 ◽

Cited By ~ 10

Author(s):

Kamal S. Gill ◽

Frank Beier ◽

Harvey A. Goldberg

Keyword(s):

Growth Plate ◽

Posttranslational Modifications ◽

Rho Kinase ◽

Cell Spreading ◽

Dynamic Structure ◽

Bone Sialoprotein ◽

Growth Plate Chondrocytes ◽

Src Signaling ◽

Rgd Sequence ◽

Specific Effects

The mammalian growth plate is a dynamic structure rich in extracellular matrix (ECM). Interactions of growth plate chondrocytes with ECM proteins regulate cell behavior. In this study, we compared chondrocyte adhesion and spreading dynamics on fibronectin (FN) and bone sialoprotein (BSP). Chondrocyte adhesion and spreading were also compared with fibroblasts to analyze potential cell-type-specific effects. Chondrocyte adhesion to BSP is independent of posttranslational modifications but is dependent on the RGD sequence in BSP. Whereas chondrocytes and fibroblasts adhered at similar levels on FN and BSP, cells displayed more actin-dependent spread on FN despite a 16× molar excess of BSP adsorbed to plastic. To identify intracellular mediators responsible for this difference in spreading, we investigated focal adhesion kinase (FAK)-Src and Rho-Rho kinase (ROCK) signaling. Although activated FAK localized to the vertices of adhered chondrocytes, levels of FAK activation did not correlate with the extent of spreading. Furthermore, Src inhibition reduced chondrocyte spreading on both FN and BSP, suggesting that FAK-Src signaling is not responsible for less cell spreading on BSP. In contrast, inhibition of Rho and ROCK in chondrocytes increased cell spreading on BSP and membrane protrusiveness on FN but did not affect cell adhesion. In fibroblasts, Rho inhibition increased fibroblast spreading on BSP while ROCK inhibition changed membrane protrusiveness of FN and BSP. In summary, we identify a novel role for Rho-ROCK signaling in regulating chondrocyte spreading and demonstrate both cell- and matrix molecule-specific mechanisms controlling cell spreading.

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The plasma membrane and matrix vesicles of mouse growth plate chondrocytes during differentiation as revealed in freeze-fracture replicas

American Journal of Anatomy ◽

10.1002/aja.1001730404 ◽

1985 ◽

Vol 173 (4) ◽

pp. 269-286 ◽

Cited By ~ 18

Author(s):

Toshitaka Akisaka ◽

Carol V. Gay

Keyword(s):

Plasma Membrane ◽

Growth Plate ◽

Freeze Fracture ◽

Matrix Vesicles ◽

Growth Plate Chondrocytes

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Expression and Functional Assessment of an Alternatively Spliced Extracellular Ca2+-Sensing Receptor in Growth Plate Chondrocytes

Endocrinology ◽

10.1210/en.2005-0256 ◽

2005 ◽

Vol 146 (12) ◽

pp. 5294-5303 ◽

Cited By ~ 55

Author(s):

Luis Rodriguez ◽

Chialing Tu ◽

Zhiqiang Cheng ◽

Tsui-Hua Chen ◽

Daniel Bikle ◽

...

Keyword(s):

Cell Surface ◽

Growth Plate ◽

Inositol Phosphate ◽

Intact Cell ◽

Chinese Hamster ◽

Full Length ◽

Wild Type ◽

Growth Plate Chondrocytes ◽

Matrix Gene ◽

Alternatively Spliced

The extracellular Ca2+-sensing receptor (CaR) plays an essential role in mineral homeostasis. Studies to generate CaR-knockout (CaR−/−) mice indicate that insertion of a neomycin cassette into exon 5 of the mouse CaR gene blocks the expression of full-length CaRs. This strategy, however, allows for the expression of alternatively spliced CaRs missing exon 5 [Exon5(−)CaRs]. These experiments addressed whether growth plate chondrocytes (GPCs) from CaR−/− mice express Exon5(−)CaRs and whether these receptors activate signaling. RT-PCR and immunocytochemistry confirmed the expression of Exon5(−)CaR in growth plates from CaR−/− mice. In Chinese hamster ovary or human embryonic kidney-293 cells, recombinant human Exon5(−)CaRs failed to activate phospholipase C likely due to their inability to reach the cell surface as assessed by intact-cell ELISA and immunocytochemistry. Human Exon5(−)CaRs, however, trafficked normally to the cell surface when overexpressed in wild-type or CaR−/− GPCs. Immunocytochemistry of growth plate sections and cultured GPCs from CaR−/− mice showed easily detectable cell-membrane expression of endogenous CaRs (presumably Exon5(−)CaRs), suggesting that trafficking of this receptor form to the membrane can occur in GPCs. In GPCs from CaR−/− mice, high extracellular [Ca2+] ([Ca2+]e) increased inositol phosphate production with a potency comparable with that of wild-type GPCs. Raising [Ca2+]e also promoted the differentiation of CaR−/− GPCs as indicated by changes in proteoglycan accumulation, mineral deposition, and matrix gene expression. Taken together, our data support the idea that expression of Exon5(−)CaRs may compensate for the loss of full-length CaRs and be responsible for sensing changes in [Ca2+]e in GPCs in CaR−/− mice.

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