Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: ERα signaling regulates MMP3 expression to induce FasL cleavage and osteoclast apoptosis.

doi: 10.1002/jbmr.1747

Figure Lengend Snippet: Fig. 3. MMP3 activity is necessary for E2-mediated cleavage of FasL. (A) U2OS-ERa cells were transfected with FASL-EGFP, then treated with vehicle control (EtOH), 10 nM E2, and/or 1 mM MMP3 inhibitor (MMP3I) for 24 hours. Cells were analyzed by flow cytometry, and the percent change in EGFP compared with EtOH-treated cells (set at 100%) is graphed. Four biological replicates are averaged. (B) U2OS-ERa cells were transfected with either an siRNA directed at luciferase (siLUC) or MMP3 (siMMP3). Forty-eight hours after transfection, cells were treated for 24 hours with vehicle control (C) or 10 nM E2. Cells were lysed and total cellular protein was immunoblotted for FasL, MMP3, and actin. (C) U2OS-ERa cells were transfected with either an siRNA directed at luciferase (siLUC) or MMP3 (siMMP3). Forty-eight hours after transfection, cells were lysed, and cDNA was analyzed by qPCR for MMP3, MMP7, and ADAM10. (D) U2OS-ERa cells were transfected with either an siRNA directed at luciferase (siLUC) or MMP3 (siMMP3). Forty-eight hours after transfection, cells were treated for 24 hours with vehicle control (C) or 10 nM E2. Cells were lysed and total cellular protein was immunoblotted for ADAM10, MMP7, and actin. Human osteoblasts (hOB) and MCF7 cells were used as controls. p < 0.05.

Article Snippet: The following antibodies were used: FasL (Santa Cruz Biotech, Santa Cruz, CA, USA; clone N-20), MMP3 (Origene, Rockville, MD, USA), MMP7 (Santa Cruz Biotech, clone MM0022-4C21), ADAM10 (Santa Cruz Biotech, clone A-3), FLAG (Sigma-Aldrich), RUNX2 (R&D Systems, Minneapolis, MN, USA) and b-actin (Sigma-Aldrich).

Techniques: Activity Assay, Transfection, Control, Flow Cytometry, Luciferase

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: ERα signaling regulates MMP3 expression to induce FasL cleavage and osteoclast apoptosis.

doi: 10.1002/jbmr.1747

Figure Lengend Snippet: Fig. 4. E2 induces MMP3 expression in primary murine osteoblasts. (A) Bone marrow–derived osteoblasts were differentiated for 2 weeks and then treated for 3 hours with vehicle control (EtOH) or 10 nM E2. MMP3 mRNA was analyzed by quantitative PCR and normalized to actin mRNA. (B) Primary calvarial osteoblasts were differentiated for 10 days and then treated for 3 hours with vehicle control (EtOH) or 10 nM E2. RNA was obtained and MMP3 mRNA was analyzed by quantitative PCR and normalized to actin mRNA. (C) Bone marrow–derived osteoblasts from wild-type (WT) and estrogen receptor knockout (ERaKO) mice were differentiated for 10 days. RNA was obtained and MMP7, ADAM10, and MMP3 mRNA was analyzed by quantitative PCR and normalized to actin mRNA. (D) Calvariae from neonatal day 2 mice were grown as organ cultures for 7 days and then treated with vehicle control (EtOH) or 10 nM E2 for 24 hours. Calvariae were fixed and paraffin-embedded. Immunofluorescence for MMP3 (green) and RUNX2 (red) was performed. (E) Quantification of MMP3 and RUNX2 immunofluorescence shown in panel D. The number of positive cells per field was counted and graphed. Error bars in all panels represent 1 SD.

Article Snippet: The following antibodies were used: FasL (Santa Cruz Biotech, Santa Cruz, CA, USA; clone N-20), MMP3 (Origene, Rockville, MD, USA), MMP7 (Santa Cruz Biotech, clone MM0022-4C21), ADAM10 (Santa Cruz Biotech, clone A-3), FLAG (Sigma-Aldrich), RUNX2 (R&D Systems, Minneapolis, MN, USA) and b-actin (Sigma-Aldrich).

Techniques: Expressing, Derivative Assay, Control, Real-time Polymerase Chain Reaction, Knock-Out, Immunofluorescence

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: ERα signaling regulates MMP3 expression to induce FasL cleavage and osteoclast apoptosis.

doi: 10.1002/jbmr.1747

Figure Lengend Snippet: Fig. 6. MMP3 activity is required for estrogen-mediated apoptosis. (A) Human monocytes were differentiated with M-CSF and RANKL to osteoclasts in a coculture with osteoblasts (MC3T3 cells) for 14 days. The cells were treated with vehicle control (EtOH) or 10 nM E2 in the presence or absence of the 50 nM MMP Inhibitor Cocktail (MMPI) or 1 mM MMP3 inhibitor (MMP3I) (B) for days 3 to 14 of differentiation. The cells were then fixed and stained for TRAP. The number of multinucleated cells was counted. Each treatment was performed in triplicate, and error bars represent the mean 1 SD. p < 0.05 E2 versus vehicle, MMPI, MMPI þ E2 or MMP3I, and MMP3I þ E2.

Article Snippet: The following antibodies were used: FasL (Santa Cruz Biotech, Santa Cruz, CA, USA; clone N-20), MMP3 (Origene, Rockville, MD, USA), MMP7 (Santa Cruz Biotech, clone MM0022-4C21), ADAM10 (Santa Cruz Biotech, clone A-3), FLAG (Sigma-Aldrich), RUNX2 (R&D Systems, Minneapolis, MN, USA) and b-actin (Sigma-Aldrich).

Techniques: Activity Assay, Control, Staining

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: ERα signaling regulates MMP3 expression to induce FasL cleavage and osteoclast apoptosis.

doi: 10.1002/jbmr.1747

Figure Lengend Snippet: Fig. 5. An MMP3 inhibitor blocks E2-mediated FasL cleavage in primary calvarial osteoblasts. Primary murine calvarial osteoblasts were differen- tiated for 10 days, followed by treatment with vehicle control (EtOH), 10 nM E2, and/or 1 mM MMP3 inhibitor for 24 hours. The conditioned media was removed and subjected to ELISA for FasL. p < 0.01 E2 versus vehicle, MMP3I and E2 þ MMP3I. MMP3I ¼ specific MMP3 inhibitor.

Article Snippet: The following antibodies were used: FasL (Santa Cruz Biotech, Santa Cruz, CA, USA; clone N-20), MMP3 (Origene, Rockville, MD, USA), MMP7 (Santa Cruz Biotech, clone MM0022-4C21), ADAM10 (Santa Cruz Biotech, clone A-3), FLAG (Sigma-Aldrich), RUNX2 (R&D Systems, Minneapolis, MN, USA) and b-actin (Sigma-Aldrich).

Techniques: Control, Enzyme-linked Immunosorbent Assay

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: ERα signaling regulates MMP3 expression to induce FasL cleavage and osteoclast apoptosis.

doi: 10.1002/jbmr.1747

Figure Lengend Snippet: Fig. 7. Model of E2 regulation of FasL transcription and cleavage. E2 increases transcription of FasL and MMP3 in osteoblasts. FasL is cleaved by MMP3 to the soluble form and then induces apoptosis of osteoclasts.

Article Snippet: The following antibodies were used: FasL (Santa Cruz Biotech, Santa Cruz, CA, USA; clone N-20), MMP3 (Origene, Rockville, MD, USA), MMP7 (Santa Cruz Biotech, clone MM0022-4C21), ADAM10 (Santa Cruz Biotech, clone A-3), FLAG (Sigma-Aldrich), RUNX2 (R&D Systems, Minneapolis, MN, USA) and b-actin (Sigma-Aldrich).

Techniques:

Journal: Glia

Article Title: MMP9 deficiency does not decrease blood–brain barrier disruption, but increases astrocyte MMP3 expression during viral encephalomyelitis

doi: 10.1002/glia.21222

Figure Lengend Snippet: MMP3 mRNA is increased in MMP9 −/− mice. A : Neutrophil elastase, MMP8, and Cathepsin G mRNA expression in brains were analyzed by real time PCR at the peak of CNS neutrophil infiltration (Day 3 and 5 p.i.) of WT and MMP9 −/− mice B . MMP3, MMP12, MMP2, and TIMP1 mRNA expression was analyzed by real time PCR in the brain of naïve, WT, and MMP9 −/− mice. * P < 0.05 C . MMP3 mRNA expression was analyzed in the brain of control and neutrophil‐depleted mice between Day 3 and 7 p.i. Data represent the average mRNA expression (±standard deviations) relative to ubiquitin of three mice per group per time point and are representative of two separate experiments.

Article Snippet: After incubation in blocking solution for 30 min, sections were incubated overnight at 4°C with goat anti‐mouse MMP3 (Santa Cruz Biotechnology, Santa Cruz, CA) and mouse anti‐mouse GFAP (Sigma).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Ubiquitin Proteomics

Journal: Glia

Article Title: MMP9 deficiency does not decrease blood–brain barrier disruption, but increases astrocyte MMP3 expression during viral encephalomyelitis

doi: 10.1002/glia.21222

Figure Lengend Snippet: MMP3 is expressed by astrocytes. A : MMP3 mRNA expression was analyzed in FACS purified populations of glial cells (CD45 − ), microglia (CD45 lo ) and CNS infiltrating leukocytes (CD45 hi ) isolated from WT and MMP9 −/− mice at Day 3 and 5 p.i. B : MMP3 and GFAP co‐expression in brain sections from MMP9 −/− mice at Day 3 p.i. Scale bar, 25 μm. C : MMP3 expression was analyzed by immunohistochemistry in WT and MMP9 −/− mice at Day 3 p.i. and at 4× higher magnification in the inset. Scale bar = 100 μm.

Article Snippet: After incubation in blocking solution for 30 min, sections were incubated overnight at 4°C with goat anti‐mouse MMP3 (Santa Cruz Biotechnology, Santa Cruz, CA) and mouse anti‐mouse GFAP (Sigma).

Techniques: Expressing, Purification, Isolation, Immunohistochemistry

Journal: Glia

Article Title: MMP9 deficiency does not decrease blood–brain barrier disruption, but increases astrocyte MMP3 expression during viral encephalomyelitis

doi: 10.1002/glia.21222

Figure Lengend Snippet: MMPs induction does not correlate with TNF and IL‐1β expression. A : Kinetics of TNF and IL‐1β mRNA expression was compared between WT and MMP9 −/− mice. B : mRNA expression of TNF, IL‐1β, MMP3, and MMP12 was analyzed at 2, 6, and 12 h after poly (I:C) injection. Data represent the average mRNA expression (±standard deviations) relative to ubiquitin of 3–4 mice per group per time point.

Article Snippet: After incubation in blocking solution for 30 min, sections were incubated overnight at 4°C with goat anti‐mouse MMP3 (Santa Cruz Biotechnology, Santa Cruz, CA) and mouse anti‐mouse GFAP (Sigma).

Techniques: Expressing, Injection, Ubiquitin Proteomics