Evaluation of VEGF-mediated signaling in primary human cells reveals a paracrine action for VEGF in osteoblast-mediated crosstalk to endothelial cells.
Communication between endothelial and bone cells is crucial for controlling vascular supply during bone growth, remodeling, and repair but the molecular mechanisms coordinating this intercellular crosstalk remain ill-defined. We have used primary human and rat long bone-derived osteoblast-like cells (HOB and LOB) and human umbilical vein endothelial cells (HUVEC) to interrogate the potential autocrine/paracrine role of vascular endothelial cell growth factor (VEGF) in osteoblast:endothelial cell (OB:EC) communication and examined whether prostaglandins (PG), known modulators of both OB and EC behavior, modify VEGF production. We found that the stable metabolite of PGI2, 6-keto-PGF(1alpha) and PGE2, induced a concentration-dependent increase in VEGF release by HOBs but not ECs. In ECs, VEGF promoted early ERK1/2 activation, late cyclooxygenase-2 (COX-2) protein induction, and release of 6-keto-PGF1alpha. In marked contrast, no significant modulation of these events was observed in HOBs exposed to VEGF, but LOBs clearly exhibited COX-dependent prostanoid release (10-fold less than EC) following VEGF treatment. A low level of osteoblast-like cell responsiveness to exogenous VEGF was supported by VEGFR2/Flk-1 immunolabelling and by blockade of VEGF-mediated prostanoid generation by a VEGFR tyrosine kinase inhibitor (TKI). HOB alkaline phosphatase (ALP) activity was increased following long-term non-contact co-culture with ECs and exposure of ECs to VEGF in this system further increased OB-like cell differentiation and markedly enhanced prostanoid release. Our studies confirm a paracrine EC-mediated effect of VEGF on OB-like cell behavior and are the first supporting a model in which prostanoids may facilitate this unidirectional VEGF-driven OB:EC communication. These findings may offer novel regimes for modulating pathological bone remodeling anomalies through the control of the closely coupled vascular supply.
Clarkin
Claire E
CE
Department of Veterinary Basic Sciences, Royal Veterinary College, University of London, London, UK. cclarkin@rvc.ac.uk
Emery
Roger J
RJ
Pitsillides
Andrew A
AA
Wheeler-Jones
Caroline P D
CP
D000174:Acromion / Q000166:cytology / Q000601:surgery; D000469:Alkaline Phosphatase / Q000032:analysis; D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D018920:Coculture Techniques; D024042:Collagen Type I / Q000032:analysis; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology; D015232:Dinoprostone / Q000032:analysis / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology; D004815:Epidermal Growth Factor / Q000378:metabolism; D011464:Epoprostenol / Q000032:analysis / Q000378:metabolism; D005260:Female; D005269:Femur / Q000166:cytology; D016650:Fluorescein-5-isothiocyanate; D019085:Fluorescent Antibody Technique, Direct; D005456:Fluorescent Dyes; D006801:Humans; D053582:Interleukin-1alpha; D009924:Organ Culture Techniques; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015675:Osteocalcin / Q000032:analysis; D019899:Paracrine Communication; D011419:Propidium; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D013997:Time Factors; D014471:Umbilical Veins / Q000166:cytology; D042461:Vascular Endothelial Growth Factor A / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
D024042:Collagen Type I; D003470:Culture Media; D005456:Fluorescent Dyes; D053582:Interleukin-1alpha; D012333:RNA, Messenger; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C467498:vascular endothelial growth factor A, rat; D015675:Osteocalcin; D011419:Propidium; D004815:Epidermal Growth Factor; D011464:Epoprostenol; D000469:Alkaline Phosphatase; D016650:Fluorescein-5-isothiocyanate; D015232:Dinoprostone