title: Characterization of proteoglycans synthesized by murine embryonal carcinoma cells (P19) reveals increased expression of perlecan (heparan sulfate proteoglycan) during neuronal differentiation.
abstract: Proteoglycans (PGs) incorporated into cell layer and secreted into media were characterized during retinoic acid-induced neuronal differentiation of cultured P19 murine embryonal carcinoma cells. Heparan sulfate significantly increased (P < 0.01) in cell layer following neuronal differentiation of P19 cells by 3.9-fold. CL-4B gel chromatography revealed the major PGs present in cell layer of stem cells eluted as a broad peak with a Kav = 0.65, and was susceptible to chondroitin ABC lyase. The chondroitin ABC lyase resistant material eluted as a broad peak between Kav = 0.40 and Kav = 0.60, and was only partially digested with heparitinase/heparinase (with resistant material eluting at Kav = 0.70). Therefore, the cell layer of stem cells contained primarily chondroitin sulfate/dermatan sulfate (CS/DS) PGs, with lesser amounts of heparan sulfate proteoglycans (HSPGs). This was confirmed by SDS-PAGE. The CS/DS PGs in the cell layer of stem cells had an apparent M(r) of approximately > 200 kDa, and the HSPGs had an apparent M(r) of approximately 140-230 kDa. In contrast, the major PGs in the cell layer of neurons consisted primarily of HSPGs, with only a minor proportion of CS/DS PGs. Furthermore, both gel filtration chromatography and SDS-PAGE analysis revealed a larger HSPG in the cell layer of neurons (Kav = 0.3-0.6 on CL-4B following chondroitin ABC lyase digestion; M(r) 170 kDa- > 400 kDa on SDS-PAGE) in comparison to stem cells (Kav = 0.4-0.6 on CL-4B following chondroitin ABC lyase digestion; M(r) 140-230 kDa on SDS-PAGE). Likewise, the major PGs secreted into media of stem cells consisted almost exclusively of CS/DS PGs, with lesser amounts of HSPGs, whereas an increase in HSPGs in the media of neurons was apparent. Western, Northern, and immunocytochemical analysis demonstrated that mRNA transcript and protein levels for a specific HSPG (i.e., perlecan) markedly increased in cell layer following P19 neuronal differentiation. Perlecan core protein was identified by Western blot analysis using specific monoclonal and polyclonal antibodies, as a large HSPG with a core protein of apparent M(r) approximately 370-400 kDa, and was observed primarily in extracts from neurons. Northern blot analysis with a cDNA to perlecan revealed a significant (P < 0.01) 12.7-fold increase in expression of perlecan in neurons (day 9) in comparison to stem cells. The increase in perlecan message during P19 neuronal differentiation was concomitant with a significant (P < 0.01) 26.3-fold increase in message for beta-amyloid precursor protein (beta PP).(ABSTRACT TRUNCATED AT 400 WORDS)
authors: Department of Pathology, University of Washington, Seattle.
authors: R T
authors: RT
authors: Sekiguchi
authors: S
authors: S
authors: Potter-Perigo
authors: K
authors: K
authors: Braun
authors: J
authors: J
authors: Miller
authors: C
authors: C
authors: Ngo
authors: K
authors: K
authors: Fukuchi
authors: T N
authors: TN
authors: Wight
authors: K
authors: K
authors: Kimata
authors: A D
authors: AD
authors: Snow
mesh_terms: D000818:Animals; D015415:Biomarkers; D015152:Blotting, Northern; D015153:Blotting, Western; D018236:Carcinoma, Embryonal / Q000378:metabolism* / Q000473:pathology; D002454:Cell Differentiation; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003470:Culture Media / Q000378:metabolism; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000378:metabolism*; D007150:Immunohistochemistry; D051379:Mice; D009474:Neurons / Q000378:metabolism / Q000473:pathology*; D010275:Parasympathetic Nervous System / Q000378:metabolism; D011509:Proteoglycans / Q000096:biosynthesis* / Q000378:metabolism*; D013234:Stem Cells / Q000378:metabolism; D013431:Sulfates / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D015415:Biomarkers; D003470:Culture Media; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D011509:Proteoglycans; D013431:Sulfates; C071927:perlecan