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Betacellulin induces angiogenesis through activation of mitogen-activated protein kinase and phosphatidylinositol 3'-kinase in endothelial cell¹

National Creative Research Initiatives Center for Endothelial Cells and Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, 790–784, Republic of Korea

³Correspondence: National Creative Research Initiatives Center for Endothelial Cells, Division of Molecular and Life Sciences, Pohang University of Science and Technology, San 31, Hyoja-Dong, Pohang, 790–784, Republic of Korea. E-mail: gykoh{at}postech.ac.kr

SPECIFIC AIM

Although betacellulin (BTC) is known as a member of the epidermal growth factor (EGF) family, its effect on angiogenesis has not been characterized. We examined the effect of BTC on angiogenic processes and their main intracellular signaling pathways in primary cultured human umbilical vein endothelial cells (HUVECs).

PRINCIPAL FINDINGS

1. BTC induces ERK1/2 and Akt phosphorylations in HUVECs
Activation of MAPK and Akt in endothelial cells plays a crucial role in angiogenesis. We measured the phosphorylation of two MAPKs, ERK1 (p44^MAPK) and ERK2 (p42^MAPK). BTC (10 ng/mL) increased ERK1/2 phosphorylation as early as 1 min and produced a maximal effect at 5 min (Fig. 1 A). The maximum mean increase in ERK2 phosphorylation was 7.9-fold. BTC (0.3–10 ng/mL) increased ERK1/2 phosphorylation in a dose-dependent manner (Fig. 1B ). Both BTC (10 ng/mL) and EGF (10 ng/mL) produced a similar level of ERK2 phosphorylation at 5 min (Fig. 1A, B ). To measure Akt activity, we measured the phosphorylation of Akt (Ser473). BTC (10 ng/mL) increased Akt phosphorylation as early as 1 min and produced a maximal effect at 5 min (Fig. 1C ). The maximum mean increase in Akt phosphorylation was 4.9-fold. BTC (0.3–10 ng/mL) increased Akt phosphorylation in a dose-dependent manner (Fig. 1D ). BTC (10 ng/mL) and EGF (10 ng/mL) produced a similar level of Akt phosphorylation at 5 min (Fig. 1C, D ).

View larger version (46K): [in this window] [in a new window]   Figure 1. BTC induces phosphorylation of ERK1/2 and Akt in HUVECs. HUVECs were incubated for 16 h in 1% serum-containing M-199 medium, then incubated with BTC or EGF(A, C) or for 5 min at the indicated concentrations (B, D). After treatment, cell lysates were harvested. Each lane contains 50 µg of total protein from cell lysates. Blots were probed with anti-phospho-ERK antibody (A, B) or anti-phospho-Akt (Ser473) antibody (C, D) (upper panels). The membrane was stripped and reprobed with anti-actin antibody or anti-Akt antibody (lower panels) to verify equal loading of protein in each lane. Fold: Densitometric analyses are presented as the relative ratio of phospho-ERK2 to actin or phospho-Akt to Akt. The relative ratio measured at time 0 or the ratio relative to control buffer (0) is arbitrarily presented as 1. Numbers represent the mean ± SD from 3 experiments. *P < 0.05 vs. time 0 or control buffer.

2. BTC-induced MAPK and Akt activations are mediated through ErbB2, ErbB3 and ErbB4 receptors in HUVECs
Our Western blot analyses indicate that HUVECs have three ErbB receptors: ErbB2, ErbB3, and ErbB4. BTC (10 ng/mL) phosphorylated all three receptors in HUVECs. These results indicate that BTC activated MAPK and Akt in HUVECs mainly through these three ErbB receptors.

3. BTC increases DNA synthesis, migration, and survival in HUVECs
BTC (10 ng/mL) increased DNA synthesis an average of 1.3-fold after 30 h treatment. After 6 h treatment, BTC (10 ng/mL) increased migration 1.4-fold. Endothelial cell survival is prerequisite for DNA synthesis and migration. Because BTC-induced activation of MAPK and phosphatidylinositol 3'-kinase (PI 3'-kinase)/Akt is often involved in survival in endothelial cells, we examined the effect of BTC on endothelial survival. Serum deprivation caused apoptosis in HUVECs. Addition of BTC (10 ng/mL) or EGF (10 ng/mL) inhibited 45–55% of serum-deprived apoptotic events. Combined pretreatment of PD98059 and wortmannin completely inhibited the BTC-induced survival effect. These results indicate that BTC exerts its antiapoptotic effect in endothelial cells through activation of MAPK and PI 3'-kinase/Akt.

4. BTC induces tube formation by endothelial cells in collagen gel
We next examined the effect of BTC on tube formation of HUVECs in type I collagen gel. After 48 h, BTC (10 ng/mL) and EGF (10 ng/mL) increased tube formation 3.7-fold and 5.5-fold, respectively. ErbB receptor inhibitor AG1478, MEK1/2 inhibitor PD98059, and PI 3'-kinase inhibitors wortmannin and LY294002 suppressed BTC-induced tube formation to varying degrees. The inhibitors alone mildly suppressed basal tube formation at 48 h. These data suggested that BTC-induced tube formation in endothelial cells is mediated through activation of ErbB receptors, PI 3'-kinase/Akt, and MAPK.

5. BTC promotes angiogenesis in vivo
To determine whether BTC is able to promote angiogenesis in vivo, we used an established in vivo angiogenesis model, the mouse Matrigel plug assay. At day 14, Matrigel containing BTC (100 ng/mL) produced more neovessels within gels than Matrigel containing control buffer (Fig. 2 A). Histochemical examination indicated that the neovessels contained red blood cells (Fig. 2B ). At day 5, Matrigel containing BTC (100 ng/mL) had more hemoglobin content than Matrigel containing control buffer (Fig. 2C ). Addition of PD98059 and wortmannin suppressed this BTC-induced angiogenic activity. Quantified by measurement of hemoglobin in the Matrigels, angiogenic activity of BTC (100 ng/mL) was comparable to that of 100 ng/mL of EGF or 20 ng/mL of VEGF (data not shown). Thus, BTC has a potent angiogenic activity in vivo through activation of MAPK and PI 3'-kinase/Akt.

View larger version (56K): [in this window] [in a new window]   Figure 2. BTC promotes neovessel formation in vivo in Matrigel plug. C57/BL6 mice were injected subcutaneously (s.c.) with 0.5 mL of Matrigel with control buffer or with BTC (100 ng) with or without PD98059 (2 µM) and wortmannin (30 nM) (PW). After 2 wk, mice were killed and Matrigel plugs were excised and fixed. A) Representative Matrigel plugs containing control buffer (CB), BTC (100 ng), or BTC (100 ng) plus PD98059 (2 µM, PD) and wortmannin (30 nM). The 3 plugs are from the same animal. B) Representative photograph of the gels shown in A cross-sectioned and stained with trichrome-Masson stain. 100x. Arrowheads indicate neovessels containing red blood cells. C) Quantification of neovessel formation by measurement of hemoglobin in the Matrigels. Eight mice were used. *P < 0.05 vs. CB; #P < 0.05 vs. BTC (10 ng/mL).

CONCLUSION AND SIGNIFICANCE
In this report, we demonstrate that BTC has a novel function as an angiogenic factor. BTC is a strong activator for intracellular MAPK and Akt in endothelial cells through ErbB2, ErbB3, and ErbB4 receptors. The activation amplitude of MAPK and Akt by BTC are comparable to that of MAPK and Akt by EGF. Accordingly, BTC promoted endothelial DNA synthesis, migration, and survival, the essential steps for angiogenesis. Indeed, BTC promoted tube formation in vitro and neovessel formation in vivo in gel plugs. Thus, we demonstrate a novel biological function of BTC in angiogenesis (Fig. 3 ) in addition to the regulatory functions already identified in pancreas and intestine.

View larger version (32K): [in this window] [in a new window]   Figure 3. Model for BTC-induced angiogenesis in endothelial cells. On HUVECs, BTC binds to the ErbB receptor types shown. Ras-dependent and -independent (?) MAPK and PI 3'-kinase/Akt pathways are activated. These pathways are the main pathways that induce survival, proliferation, migration and tube formation. These increased cellular processes of endothelial cells promote angiogenesis in vivo.

Activation of MAPK and PI 3'-kinase/Akt in endothelial cells is a crucial intracellular signaling step for angiogenesis. Growth factors bind to their receptor tyrosine kinases in endothelial cells and induce angiogenesis through activation of these kinases. Our Western blot analyses indicate that primary cultured HUVECs express ErbB2, ErbB3, and ErbB4, but not ErbB1. Our biochemical results indicate that BTC induces phosphorylation of ErbB2, ErbB3, and ErbB4. Moreover, BTC-induced phosphorylation of MAPK and Akt is not suppressed by a selectively inhibitory concentration of ErbB1 receptor. Therefore, BTC-induced activation of MAPK and Akt could occur through activation of ErbB4 homodimer and ErbB2/ErbB3 or ErbB2/ErbB4 heterodimers (Fig. 3) .

Angiogenesis is an essential process in tumor–host interactions for tumor growth, maintenance, and metastasis. Because recent reports indicate that EGF receptors are closely involved in tumor angiogenesis through induction of angiogenic molecules such as VEGF and angiopoietin-1, BTC, in addition to having a direct angiogenic role, may also act as an indirect angiogenic factor through induction of angiogenic molecules in vivo. Like other cancers, pancreatic cancer has an extensive interaction with host cells for progressive growth and metastasis. BTC expression is elevated 7-fold in 9 of 10 pancreatic cancers compared to normal pancreas. Expression of EGF receptors is enhanced in pancreatic cancers. BTC would be not only a significant growth factor for pancreatic cancer cells, but also a strong angiogenic growth factor and endothelial cell survival factor. As expected, most pancreatic cancer is characterized by highly angiogenic, extensive local invasion and early hematogenous metastasis. In fact, a blockade of EGF receptors using the corresponding kinase inhibitor significantly inhibited growth and metastasis of human pancreatic cancer growing orthotopically in nude mice. The therapeutic effect was mediated in part by inhibition of tumor angiogenesis and increased apoptosis of tumor-associated endothelial cells. Furthermore, a blockade of EGF receptors using a blocking antibody or its kinase inhibitor has emerged as a novel approach in the treatment of cancer. It would be important to clarify the interactions among pancreatic cancer, BTC, angiogenesis, and EGFR blockade for future studies.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0570fje; to cite this article, use FASEB J. (December 3, 2002) 10.1096/fj.02-0570fje

² These two authors contributed equally to this work.

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