Questioning the reliability of p-nitrophenyl-β- d-xyloside as probe to study the metabolic effects of abrogated proteoglycan synthesis in cultured cells

Gressner, A. M.

doi:10.1016/0006-2952(91)90599-z

Cited by 4 publications

(3 citation statements)

References 38 publications

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“…Alternatively, previous studies (23,46) have shown that MU and pNP derivatives of xylosides, MU-Xyl and pNPXyl, significantly inhibited the biosynthesis of HA as well as sulfated glycosaminoglycans. As for the mechanism, it was suggested that free pNP, which is generated by the enzymatic hydrolysis of pNP-Xyl, mediated the inhibitory effect (47). In the current study, we also detected a significant liberation of pNP from its sugar derivatives.…”

Section: Fig 7 Enhanced Inhibition Of Has Activity By Recombinant Usupporting

confidence: 67%

A Novel Mechanism for the Inhibition of Hyaluronan Biosynthesis by 4-Methylumbelliferone

Kakizaki

Kojima

Takagaki

et al. 2004

Journal of Biological Chemistry

246

213

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Specific inhibitors of hyaluronan (HA) biosynthesis can be valuable therapeutic agents to prevent cancer invasion and metastasis. We have found previously that 4-methylumbelliferone (MU) inhibits HA synthesis in human skin fibroblasts and in group C Streptococcus. In this paper, the inhibition mechanism in mammalian cells was investigated using rat 3Y1 fibroblasts stably expressing HA synthase (HAS) 2. Exposure of the transfectants to the inhibitor resulted in significant reduction of HA biosynthesis and matrix formation. The evaluation of HAS transcripts and analysis of cell-free HA synthesis demonstrated the post-transcriptional suppression of HAS activity by MU. Most interesting, the post-transcriptional suppression of HAS activity was also observed using p-nitrophenol, a well known substrate for UDP-glucuronyltransferases (UGT). We investigated whether the inhibition was exerted by the glucuronidation of MU using both high pressure liquid chromatography and TLC analyses. The production of MU-glucuronic acid (GlcUA) was consistent with the inhibition of HA synthesis in HAS transfectants. MU-GlcUA was also detected at a similar level in control cells, suggesting that the glucuronidation was mediated by an endogenous UGT. Elevated levels of UGT significantly enhanced the inhibitory effects of MU. In contrast, the inhibition by MU was diminished to the control level when an excess of UDP-GlcUA was added to the cell-free HA synthesis system. We propose a novel mechanism for the MU-mediated inhibition of HA synthesis involving the glucuronidation of MU by endogenous UGT resulting in a depletion of UDP-GlcUA.

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Section: Fig 7 Enhanced Inhibition Of Has Activity By Recombinant Usupporting

confidence: 67%

A Novel Mechanism for the Inhibition of Hyaluronan Biosynthesis by 4-Methylumbelliferone

Kakizaki

Kojima

Takagaki

et al. 2004

Journal of Biological Chemistry

246

213

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“…Two recently described methodologic problems with the use of PNP-xylosides were encountered. First, PNP has been shown to interfere with the cytoskeleton (Gressner, 1991). Concentrations of PNP between 100 and 500 FM caused a flattened appearance of vertically sectioned epithelia.…”

Section: Discussionmentioning

confidence: 99%

“…When these cultures were sectioned and stained, both treated and untreated cultures were stratified. Subtle changes in the epithelial organization sometimes appeared in cultures incubated with high concentrations of PNP-xyloside, but it has been reported that PNP alone can disrupt the organization of cells (Gressner, 1991).…”

Section: Proteoglycan Disruption Had Little Effect Onmentioning

confidence: 99%

Response of stratified cultures of human keratinocytes to disruption of proteoglycan synthesis by p‐nitrophenyl‐β‐D‐xylopyranoside

Haggerty

Bretton

Milstone

1994

Journal Cellular Physiology

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Proteoglycans play a role in regulating proliferation and adhesion of cells to each other and to the basal lamina. Synthesis of proteoglycans is disrupted by beta-xylosides, which serve as alternate substrate sites for glycosaminoglycan chain attachment and therefore prevent glycosylation of the core protein. We have investigated the effects of p-nitrophenyl-beta-D-xylopyranoside (PNP-xyloside) on cultured human keratinocytes. Stratified cultures were incubated for 7 days with PNP-xyloside (0.05-2.0 mM). Concentrations as low as 0.05 mM increased the secretion of free chondroitin sulfate by 10-15-fold over untreated cultures. Cell-associated proteoglycan decreased as PNP-xyloside concentration increased. At 2 mM PNP-xyloside, heparan sulfate as well as chondroitin sulfate addition to core proteins was disrupted: the core protein of epican, a heparan sulfate form of CD44 found on keratinocytes, was detected immunologically but lacked heparan sulfate. 2.0 mM PNP-xyloside reduced the number of attached cells by 20-25% after 7 days, but had little effect on morphology or protein synthesis. These results indicate that intact proteoglycans are not critical for maintaining epidermal keratinocyte stratification, cell-cell adhesion, or growth.

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alpha- and beta-xylosides modulate the syunthesis of fibronectin and thrombospondin-1 by endothelial cells

Canfield¹,

Sutton²,

Hiscock³

et al. 1994

Biochimica et Biophysica Acta (BBA) - General Subjects

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Questioning the reliability of p-nitrophenyl-β- d-xyloside as probe to study the metabolic effects of abrogated proteoglycan synthesis in cultured cells

Cited by 4 publications

References 38 publications

A Novel Mechanism for the Inhibition of Hyaluronan Biosynthesis by 4-Methylumbelliferone

A Novel Mechanism for the Inhibition of Hyaluronan Biosynthesis by 4-Methylumbelliferone

Response of stratified cultures of human keratinocytes to disruption of proteoglycan synthesis by p‐nitrophenyl‐β‐D‐xylopyranoside

alpha- and beta-xylosides modulate the syunthesis of fibronectin and thrombospondin-1 by endothelial cells

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