Role of PG-M proteoglycan and hyaluronic acid that accompany the differentiation of 3T3-L1 preadipocytes with isobutylmethylxanthine, dexamethasone and insulin.

CYNTHIA F ZIZOLA; VANINA JULIANELLI; GABRIEL BERTOLESI; MASAKI YANAGISHITA; JUAN CARLOS CALVO

MATRIX BIOLOGY

Año: 2007 vol. 26 p. 419 - 430

0945-053X

Abstract We have previously shown that during the adipose conversion of these cells the culture medium changed its viscoelastic properties due to the presence of hyaluronan and a chondroitin sulfate proteoglycan [Calvo, J.C., Rodbard, D., Katki, A., Chernick, S., and Yanagishita, M., 1991. Differentiation of 3T3-L1 preadipocytes with 3-isobutyl-1-methylxanthine and dexamethasone stimulates cell-associated and soluble chondroitin 4-sulfate proteoglycans. J. Biol. Chem. 266, 11237–11244., Calvo, J.C., Gandjbakhche, A.H., Nossal, R., Hascall, V.C., and Yanagishita, M., 1993. Rheological effects of the presence of hyaluronic acid in the extracellular media of differentiated 3T3-L1 preadipocyte cultures. Arch. Biochem. Biophys. 302, 468–475]. Here, we analyze the time course for the appearance of these molecules during drug-induced cell differentiation. The synthesis of both hyaluronan and the proteoglycan, was maximal at 48 h in the presence of isobutylmethylxanthine and dexamethasone, but while hyaluronan remained high after changing the culture medium, the proteoglycan dropped to almost basal levels after a few days. Northern analysis revealed the presence of message for a “versican-like” molecule as well as the possibility of alternative splicing. Three major bands of 9.39, 8.48, and 7.69 kb appeared in the analysis. These bands showed a dramatic increase in intensity when RNA from nondifferentiated cells was compared to differentiating 3T3-L1 cells. In addition, when the time course of appearance for this message was analyzed, it perfectly correlated the metabolic labeling of the glycosaminoglycans during cell culture. The nucleotide sequencing of two exons revealed between a 100–94% homology with proteoglycan PG-M from murine endothelial cells. At least 13% of the proteoglycan was able to bind hyaluronan. Disruption of the synthesis of the proteoglycan molecule by exogenous addition of xyloside, did not prevent triglyceride accumulation but was inhibitory to preconfluent 3T3-L1 cell proliferation. Coating of plastic culture dishes with conditioned medium from differentiating 3T3-L1 cells, resulted in decreased cell adhesion. Cell adhesion was partially recovered after degradation of hyaluronan and chondroitin sulfate by enzymatic treatment. All these results indicate a possible role of these molecules in the observed changes in the viscoelastic properties of the culture medium, as well as open the field for a more thorough study of their role in 3T3-L1 cell proliferation and/or differentiation.–11244., Calvo, J.C., Gandjbakhche, A.H., Nossal, R., Hascall, V.C., and Yanagishita, M., 1993. Rheological effects of the presence of hyaluronic acid in the extracellular media of differentiated 3T3-L1 preadipocyte cultures. Arch. Biochem. Biophys. 302, 468–475]. Here, we analyze the time course for the appearance of these molecules during drug-induced cell differentiation. The synthesis of both hyaluronan and the proteoglycan, was maximal at 48 h in the presence of isobutylmethylxanthine and dexamethasone, but while hyaluronan remained high after changing the culture medium, the proteoglycan dropped to almost basal levels after a few days. Northern analysis revealed the presence of message for a “versican-like” molecule as well as the possibility of alternative splicing. Three major bands of 9.39, 8.48, and 7.69 kb appeared in the analysis. These bands showed a dramatic increase in intensity when RNA from nondifferentiated cells was compared to differentiating 3T3-L1 cells. In addition, when the time course of appearance for this message was analyzed, it perfectly correlated the metabolic labeling of the glycosaminoglycans during cell culture. The nucleotide sequencing of two exons revealed between a 100–94% homology with proteoglycan PG-M from murine endothelial cells. At least 13% of the proteoglycan was able to bind hyaluronan. Disruption of the synthesis of the proteoglycan molecule by exogenous addition of xyloside, did not prevent triglyceride accumulation but was inhibitory to preconfluent 3T3-L1 cell proliferation. Coating of plastic culture dishes with conditioned medium from differentiating 3T3-L1 cells, resulted in decreased cell adhesion. Cell adhesion was partially recovered after degradation of hyaluronan and chondroitin sulfate by enzymatic treatment. All these results indicate a possible role of these molecules in the observed changes in the viscoelastic properties of the culture medium, as well as open the field for a more thorough study of their role in 3T3-L1 cell proliferation and/or differentiation.–475]. Here, we analyze the time course for the appearance of these molecules during drug-induced cell differentiation. The synthesis of both hyaluronan and the proteoglycan, was maximal at 48 h in the presence of isobutylmethylxanthine and dexamethasone, but while hyaluronan remained high after changing the culture medium, the proteoglycan dropped to almost basal levels after a few days. Northern analysis revealed the presence of message for a “versican-like” molecule as well as the possibility of alternative splicing. Three major bands of 9.39, 8.48, and 7.69 kb appeared in the analysis. These bands showed a dramatic increase in intensity when RNA from nondifferentiated cells was compared to differentiating 3T3-L1 cells. In addition, when the time course of appearance for this message was analyzed, it perfectly correlated the metabolic labeling of the glycosaminoglycans during cell culture. The nucleotide sequencing of two exons revealed between a 100–94% homology with proteoglycan PG-M from murine endothelial cells. At least 13% of the proteoglycan was able to bind hyaluronan. Disruption of the synthesis of the proteoglycan molecule by exogenous addition of xyloside, did not prevent triglyceride accumulation but was inhibitory to preconfluent 3T3-L1 cell proliferation. Coating of plastic culture dishes with conditioned medium from differentiating 3T3-L1 cells, resulted in decreased cell adhesion. Cell adhesion was partially recovered after degradation of hyaluronan and chondroitin sulfate by enzymatic treatment. All these results indicate a possible role of these molecules in the observed changes in the viscoelastic properties of the culture medium, as well as open the field for a more thorough study of their role in 3T3-L1 cell proliferation and/or differentiation.“versican-like” molecule as well as the possibility of alternative splicing. Three major bands of 9.39, 8.48, and 7.69 kb appeared in the analysis. These bands showed a dramatic increase in intensity when RNA from nondifferentiated cells was compared to differentiating 3T3-L1 cells. In addition, when the time course of appearance for this message was analyzed, it perfectly correlated the metabolic labeling of the glycosaminoglycans during cell culture. The nucleotide sequencing of two exons revealed between a 100–94% homology with proteoglycan PG-M from murine endothelial cells. At least 13% of the proteoglycan was able to bind hyaluronan. Disruption of the synthesis of the proteoglycan molecule by exogenous addition of xyloside, did not prevent triglyceride accumulation but was inhibitory to preconfluent 3T3-L1 cell proliferation. Coating of plastic culture dishes with conditioned medium from differentiating 3T3-L1 cells, resulted in decreased cell adhesion. Cell adhesion was partially recovered after degradation of hyaluronan and chondroitin sulfate by enzymatic treatment. All these results indicate a possible role of these molecules in the observed changes in the viscoelastic properties of the culture medium, as well as open the field for a more thorough study of their role in 3T3-L1 cell proliferation and/or differentiation.–94% homology with proteoglycan PG-M from murine endothelial cells. At least 13% of the proteoglycan was able to bind hyaluronan. Disruption of the synthesis of the proteoglycan molecule by exogenous addition of xyloside, did not prevent triglyceride accumulation but was inhibitory to preconfluent 3T3-L1 cell proliferation. Coating of plastic culture dishes with conditioned medium from differentiating 3T3-L1 cells, resulted in decreased cell adhesion. Cell adhesion was partially recovered after degradation of hyaluronan and chondroitin sulfate by enzymatic treatment. All these results indicate a possible role of these molecules in the observed changes in the viscoelastic properties of the culture medium, as well as open the field for a more thorough study of their role in 3T3-L1 cell proliferation and/or differentiation.