A novel endothelial-specific heat shock protein HspA12B is required in both zebrafish development and endothelial functions in vitro.
A zebrafish transcript dubbed GA2692 was initially identified via a whole-mount in situ hybridization screen for vessel specific transcripts. Its mRNA expression during embryonic development was detected in ventral hematopoietic and vasculogenic mesoderm and later throughout the vasculature up to 48 hours post fertilization. Morpholino-mediated knockdown of GA2692 in embryos resulted in multiple defects in vasculature, particularly, at sites undergoing active capillary sprouting: the intersegmental vessels, sub-intestinal vessels and the capillary sprouts of the pectoral fin vessel. During the course of these studies, a homology search indicated that GA2692 is the zebrafish orthologue of mammalian HspA12B, a distant member of the heat shock protein 70 (Hsp70) family. By a combination of northern blot and real-time PCR analysis, we showed that HspA12B is highly expressed in human endothelial cells in vitro. Knockdown of HspA12B by small interfering RNAs (siRNAs) in human umbilical vein endothelial cells blocked wound healing, migration and tube formation, whereas overexpression of HspA12B enhanced migration and accelerated wound healing - data that are consistent with the in vivo fish phenotype obtained in the morpholino-knockdown studies. Phosphorylation of Akt was consistently reduced by siRNAs against HspA12B. Overexpression of a constitutively active form of Akt rescued the inhibitory effects of knockdown of HspA12B on migration of human umbilical vein endothelial cells. Collectively, our data suggests that HspA12B is a highly endothelial-cell-specific distant member of the Hsp70 family and plays a significant role in endothelial cells during development and angiogenesis in vitro, partially attributable to modulation of Akt phosphorylation.
Hu
Guang
G
Renal Division, Center for Study of the Tumor Microenvironment and Center for Vascular Biology Research, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA.
Tang
Jian
J
Zhang
Bo
B
Lin
Yanfeng
Y
Hanai
Jun-Ichi
J
Galloway
Jenna
J
Bedell
Victoria
V
Bahary
Nathan
N
Han
Zhihua
Z
Ramchandran
Ramani
R
Thisse
Bernard
B
Thisse
Christine
C
Zon
Leonard I
LI
Sukhatme
Vikas P
VP
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002465:Cell Movement / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D017023:Coronary Angiography / Q000379:methods; D004625:Embryo, Nonmammalian; D047108:Embryonic Development / Q000235:genetics; D042783:Endothelial Cells / Q000378:metabolism; D004730:Endothelium, Vascular / Q000502:physiology*; D018507:Gene Expression Regulation, Developmental; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics* / Q000502:physiology; D006360:Heat-Shock Proteins / Q000235:genetics / Q000502:physiology*; D006801:Humans; D018919:Neovascularization, Physiologic / Q000235:genetics; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D051058:Oncogene Protein v-akt / Q000378:metabolism; D010766:Phosphorylation; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000009:adverse effects; D014162:Transfection; D015027:Zebrafish / Q000196:embryology* / Q000502:physiology; D029961:Zebrafish Proteins / Q000235:genetics / Q000502:physiology*
D018840:HSP70 Heat-Shock Proteins; C472131:HSPA12B protein, human; D006360:Heat-Shock Proteins; C515822:HspA12B protein, zebrafish; D016376:Oligonucleotides, Antisense; D034741:RNA, Small Interfering; D029961:Zebrafish Proteins; D051058:Oncogene Protein v-akt; D000469:Alkaline Phosphatase