CHARACTERIZATION OF PROTEOGLYCANS SYNTHESIZED BY MURINE EMBRYONAL CARCINOMA-CELLS (P19) REVEALS INCREASED EXPRESSION OF PERLECAN (HEPARAN-SULFATE PROTEOGLYCAN) DURING NEURONAL DIFFERENTIATION

Proteoglycans (PGs) incorporated into cell layer and secreted into med ia were characterized during retinoic acid-induced neuronal differenti ation of cultured P19 murine embryonal carcinoma cells. Heparan sulfat e significantly increased (P < 0.01) in cell layer following neuronal differentiation of P19 cells by 3.9-fold. CL-4B gel chromatography rev ealed the major PGs present in cell layer of stem cells eluted as a br oad peak with a K-av = 0.65, and was susceptible to chondroitin ABC ly ase. The chondroitin ABC lyase resistant material eluted as a broad pe ak between K-av = 0.40 and K-av = 0.60, and was only partially digeste d with heparitinase/heparinase (with resistant material eluting at K-a v = 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-P AGE. The CS/DS PGs in the cell layer of stem cells had an apparent M(r ) of similar to >200 kDa, and the HSPGs had an apparent M(r) of simila r to 140-230 kDa. In contrast, the major PGs in the cell layer of neur ons consisted primarily of HSPGs, with only a minor proportion of CS/D S PGs. Furthermore, both gel filtration chromatography and SDS-PAGE an alysis revealed a larger HSPG in the cell layer of neurons (K-av = 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 (K-av = 0.4-0.6 on C L-4B following chondroitin ABC lyase digestion; M(r) 140-230 kDa on SD S-PAGE). Likewise, the major PGs secreted into media of stem cells con sisted almost exclusively of CS/DS PGs, with lesser amounts of HSPGs, whereas an increase in HSPGs in the media of neurons was apparent. Wes tern, Northern, and immunocytochemical analysis demonstrated that mRNA transcript and protein levels for a specific HSPG (i.e., perlecan) ma rkedly increased in cell layer following P19 neuronal differentiation. Perlecan core protein was identified by Western blot analysis using s pecific monoclonal and polyclonal antibodies, as a large HSPG with a c ore protein of apparent M(r) similar to 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 e xpression of perlecan in neurons (day 9) in comparison to stem cells. The increase in perlecan message during P19 neuronal differentiation w as concomitant with a significant (P < 0.01) 26.3-fold increase in mes sage for beta-amyloid precursor protein (beta PP). Immunohistochemical staining of P19 cultures with perlecan-specific antibodies revealed p erlecan primarily localized to cell bodies and neurites of differentia ted P19 cells which were identified as neurons on adjacent sections by positive immunostaining with neuronal markers (choline acetyltransfer ase and acetyl cholinesterase). This study demonstrates for the first time that perlecan is synthesized by neuron-like cells and will serve as a baseline for future studies utilizing the P19 cell culture system to assess the influence of specific PGs/ GAGs on beta PP metabolism. (C) 1994 Wiley-Liss, Inc.