Human α1 type IV collagen NC1 domain exhibits distinct antiangiogenic activity mediated by α1β1 integrin
Akulapalli Sudhakar, … , Dominic Cosgrove, Raghu Kalluri
Akulapalli Sudhakar, … , Dominic Cosgrove, Raghu Kalluri
Published October 3, 2005
Citation Information: J Clin Invest. 2005;115(10):2801-2810. https://doi.org/10.1172/JCI24813.
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Research Article Angiogenesis

Human α1 type IV collagen NC1 domain exhibits distinct antiangiogenic activity mediated by α1β1 integrin

Abstract

Human noncollagenous domain 1 of the α1 chain of type IV collagen [α1(IV)NC1], or arresten, is derived from the carboxy terminal of type IV collagen. It was shown to inhibit angiogenesis and tumor growth in vivo; however, the mechanisms involved are not known. In the present study we demonstrate that human α1(IV)NC1 binds to α1β1 integrin, competes with type IV collagen binding to α1β1 integrin, and inhibits migration, proliferation, and tube formation by ECs. Also, α1(IV)NC1 pretreatment inhibited FAK/c-Raf/MEK/ERK1/2/p38 MAPK activation in ECs but had no effect on the PI3K/Akt pathway. In contrast, α1(IV)NC1 did not affect proliferation, migration, or the activation of FAK/c-Raf/MEK1/2/p38/ERK1 MAPK pathway in α1 integrin receptor knockout ECs. Consistent with this, α1(IV)NC1 elicited significant antiangiogenic effects and tumor growth inhibition in vivo but failed to do the same in α1 integrin receptor knockout mice. This suggests a highly specific, α1β1 integrin–dependent antiangiogenic activity of α1(IV)NC1. In addition, α1(IV)NC1 inhibited hypoxia-induced expression of hypoxia-inducible factor 1α and VEGF in ECs cultured on type IV collagen by inhibiting ERK1/2 and p38 activation. This unravels a hitherto unknown function of human α1(IV)NC1 and suggests a critical role for integrins in hypoxia and hypoxia-induced angiogenesis. Collectively, the above data indicate that α1(IV)NC1 is a potential therapeutic candidate for targeting tumor angiogenesis.

Authors

Akulapalli Sudhakar, Pia Nyberg, Venkateshwar G. Keshamouni, Arjuna P. Mannam, Jian Li, Hikaru Sugimoto, Dominic Cosgrove, Raghu Kalluri

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Figure 6

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Effect of α1(IV)NC1 on hypoxia-induced HIF-1α and VEGF expression. Serum...
Effect of α1(IV)NC1 on hypoxia-induced HIF-1α and VEGF expression. Serum-starved WT MLECs were treated with α1(IV)NC1 or PD98059/SB203580 in complete medium, plated on type IV collagen–coated plates, and then exposed to hypoxic conditions for 12 and 24 hours. Total cytosolic cell extracts were prepared and analyzed by SDS-PAGE and Western blot with different antibodies shown in the figure. (A) Hypoxia-induced ERK1/2 phosphorylation upon α1(IV)NC1 treatment (A, top panel) and total ERK1/2 protein (A, bottom panel). (B) Hypoxia-induced p38 phosphorylation upon α1(IV)NC1 treatment (B, top panel) and total p38 protein (B, bottom panel). (C) Hypoxia-induced expression of HIF-1α and VEGF proteins upon α1(IV)NC1 treatment (C, top and middle panels); actin levels are shown as an internal control (C, bottom panel). (D) Hypoxia-induced expression of HIF-1α and VEGF proteins upon α1(IV)NC1 treatment in α1 integrin–/– MLECs (D, top and middle panels); actin levels are shown as an internal control (D, bottom panel). (E–G) Hypoxia-induced MEK1/2 and p38 phosphorylation and HIF-1α and VEGF proteins upon PD98059 (50 μM)/SB203580 (20 μM) treatment, similarly as shown in A–C.