mouse anti-integrin α9β1  (Millipore)

Journal: The Journal of Cell Biology

Article Title: EMILIN1–α4/α9 integrin interaction inhibits dermal fibroblast and keratinocyte proliferation

doi: 10.1083/jcb.201008013

Figure Lengend Snippet: The EMILIN1 gC1q domain inhibits cell proliferation through the interaction with the α4 and α9 integrin subunit. (A) Proliferation of sarcoma (HT1080 and RD), carcinoma (HeLa and CaCo-2), and immortal keratinocyte (HaCaT) cell lines in the presence or in the absence of 50 µg/ml of soluble gC1q added to the culture medium for 24 h. The percentage of mean values (±SD) of the number of BrdU-positive cells per field of three independent experiments is reported. *, P < 0.05. (B) FACS analysis of α4 and α9 integrin subunit expression levels in HT1080, RD, CaCo-2, HeLa, and HaCaT cells. (C) Cell adhesion of HT1080 and CaCo-2 cells to gC1q. The cells were preincubated with anti–α4 integrin subunit mAb (P1H4), anti–α9 integrin subunit mAb (Y9A2), or anti–β integrin subunit mAb (4B4; final concentration, 10 µg/ml) for 15 min at 37°C and were then allowed to adhere at 37°C for 20 min. Data are expressed as the means ± SD of three independent experiments with six replicates. *, P < 0.05; **, P < 0.001. (D–F) Proliferation inhibition of HT1080, CaCo-2, and HaCaT cells expressed as the percentage versus the respective control (ctrl). The gC1q domain was used at a concentration of 5 µg/ml; the monoclonal antibody anti-gC1q (1H2) and the function blocking monoclonal antibodies anti–α4 integrin subunit (P1H4) and anti–α9 integrin subunit (Y9A2) were used at 10 µg/ml. Data are expressed as the means ± SD of three independent experiments. *, P < 0.05; **, P < 0.001. (G) Effect of gC1q and the mutants E933A, G945A, and the deleted form on CaCo-2 cell proliferation monitored using the XCELLigence system. The cell index after 48 h of dynamic monitoring calculated as the mean ± SD from n = 3 experiments with n = 6 replicates is reported. *, P < 0.001. (H) Representative immunofluorescence images of skin cryostat sections of 7-wk-old WT mice stained for EMILIN1 and for the α9 integrin subunit. Bars, 25 µm.

Article Snippet: Moreover, mouse anti-CdK2 (BD), mouse anti–Cyclin D1/2 (Millipore), rabbit anti–mature TGF-β1 (BioVision Research Products), rabbit anti–pan-CK antibody (Dako), mouse anti–integrin α9β1 (clone Y9A2) and anti–integrin α4 (clone P1H4; Millipore), goat anti–mouse integrin α9β1 (LifeSpan BioSciences), and mouse anti–β1 integrin subunit (clone 4B4; Beckman Coulter) antibodies were used.

Techniques: Expressing, Concentration Assay, Inhibition, Blocking Assay, Immunofluorescence, Staining

Journal: The Journal of Cell Biology

Article Title: EMILIN1–α4/α9 integrin interaction inhibits dermal fibroblast and keratinocyte proliferation

doi: 10.1083/jcb.201008013

Figure Lengend Snippet: Proposed model for the regulatory role of EMILIN1 in skin homeostasis. The illustration summarizes the proposed molecular mechanism underlying the regulatory role of EMILIN1 in skin proliferation. (A) TGF-β triggers cytostatic signal pathways mainly through pSmad2 (Ser465/467) activation and modulates PI3K/Akt signaling by regulating PTEN expression. showed that EMILIN1 inhibits TGF-β processing by binding specifically to the pro–TGF-β precursor and by preventing its maturation in the extracellular space. Here, we demonstrated that EMILIN1 binding to dermal fibroblast and basal keratinocytes α4β1/α9β1 integrins empowers the down-regulation of proliferative cues induced by TGF-β. This effect is mediated by α4/α9β1-dependent PTEN activation and inhibition of pErk1/2 proproliferative activity. (B) The increased levels of mature TGF-β and the lack of α4/α9β1 integrin–specific engagement by the lack of EMILIN1 result in PTEN down-regulation and, hence, reduced activity. This determines the activation of proliferative pathways such as pAkt and pErk1/2 that in turn lead to a reduction of TGF-β signaling via increased Erk1/2-dependent phosphorylation of Smad2 at inhibitory Ser245/250/255. In conclusion, we provide the first evidence for the central role of PTEN in the cross talk between α4/α9β1 integrin and TGF-β signal pathways.

Article Snippet: Moreover, mouse anti-CdK2 (BD), mouse anti–Cyclin D1/2 (Millipore), rabbit anti–mature TGF-β1 (BioVision Research Products), rabbit anti–pan-CK antibody (Dako), mouse anti–integrin α9β1 (clone Y9A2) and anti–integrin α4 (clone P1H4; Millipore), goat anti–mouse integrin α9β1 (LifeSpan BioSciences), and mouse anti–β1 integrin subunit (clone 4B4; Beckman Coulter) antibodies were used.

Techniques: Activation Assay, Expressing, Binding Assay, Inhibition, Activity Assay

Journal: The Journal of Cell Biology

Article Title: EMILIN1–α4/α9 integrin interaction inhibits dermal fibroblast and keratinocyte proliferation

doi: 10.1083/jcb.201008013

Figure Lengend Snippet: Proposed model for the regulatory role of EMILIN1 in skin homeostasis. The illustration summarizes the proposed molecular mechanism underlying the regulatory role of EMILIN1 in skin proliferation. (A) TGF-β triggers cytostatic signal pathways mainly through pSmad2 (Ser465/467) activation and modulates PI3K/Akt signaling by regulating PTEN expression. showed that EMILIN1 inhibits TGF-β processing by binding specifically to the pro–TGF-β precursor and by preventing its maturation in the extracellular space. Here, we demonstrated that EMILIN1 binding to dermal fibroblast and basal keratinocytes α4β1/α9β1 integrins empowers the down-regulation of proliferative cues induced by TGF-β. This effect is mediated by α4/α9β1-dependent PTEN activation and inhibition of pErk1/2 proproliferative activity. (B) The increased levels of mature TGF-β and the lack of α4/α9β1 integrin–specific engagement by the lack of EMILIN1 result in PTEN down-regulation and, hence, reduced activity. This determines the activation of proliferative pathways such as pAkt and pErk1/2 that in turn lead to a reduction of TGF-β signaling via increased Erk1/2-dependent phosphorylation of Smad2 at inhibitory Ser245/250/255. In conclusion, we provide the first evidence for the central role of PTEN in the cross talk between α4/α9β1 integrin and TGF-β signal pathways.

Article Snippet: Moreover, mouse anti-CdK2 (BD), mouse anti–Cyclin D1/2 (Millipore), rabbit anti–mature TGF-β1 (BioVision Research Products), rabbit anti–pan-CK antibody (Dako), mouse anti–integrin α9β1 (clone Y9A2) and anti–integrin α4 (clone P1H4; Millipore), goat anti–mouse integrin α9β1 (LifeSpan BioSciences), and mouse anti–β1 integrin subunit (clone 4B4; Beckman Coulter) antibodies were used.

Techniques: Activation Assay, Expressing, Binding Assay, Inhibition, Activity Assay, Phospho-proteomics