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Hepatocyte DNA replication is abolished by inhibitors selecting protein phosphatase 2A rather than phosphatase 1
G Mellgren1, O K Vintermyr, R Bøe
1Department of Anatomy, University of Bergen, Norway.
Abstract:
Primary rat hepatocytes exposed to the phosphoprotein phosphatase (PP) inhibitors microcystin-LR and okadaic acid showed extensive surface protrusions and release of cell fragments, like cells in apoptosis. Microinjected microcystin fully reproduced these effects; the calculated intracellular concentration required for 50% effect being about 1 microM. The effects were counteracted by antagonists of calmodulin or of the multifunctional calmodulin-activated protein kinase II. The DNA replication of the epidermal growth factor-stimulated hepatocytes was nearly completely inhibited by okadaic acid at concentrations below those giving overt morphological effects. However, microcystin did not inhibit the DNA replication. Calmodulin antagonists counteracted the effect of okadaic acid on DNA replication. Microinjection of inhibitor-1 and inhibitor-2 (both directed against PP1) had no effect on DNA replication. Based on the known selectivity of okadaic acid for PP type 2A versus that of type 1, and the lack of such selectivity for microcystin, it is concluded that DNA replication is abolished by moderate inhibition of PP2A. Inhibition of PP1 did not impede DNA replication, suggesting that the two major liver phosphatases may have opposite roles in the regulation of hepatocyte DNA replication.
Insights
Phosphoprotein phosphatase (PP) inhibitors like okadaic acid disrupt hepatocyte structure and inhibit DNA replication by targeting PP2A. Microcystin-LR affects cell morphology but not DNA replication, suggesting distinct phosphatase roles.
Area of Science:
- Hepatocyte biology
- Cell signaling
- Molecular toxicology
Background:
- Phosphoprotein phosphatases (PPs) are crucial enzymes regulating cellular processes.
- PP inhibitors like microcystin-LR and okadaic acid are widely used to study phosphatase function.
- Hepatocyte proliferation is a tightly regulated process involving complex signaling pathways.
Purpose of the Study:
- To investigate the effects of PP inhibitors on primary rat hepatocytes.
- To determine the role of specific phosphatases (PP1 and PP2A) in regulating hepatocyte DNA replication and morphology.
- To elucidate the signaling pathways involved in PP inhibitor-induced cellular changes.
Main Methods:
- Primary rat hepatocytes were treated with microcystin-LR and okadaic acid.
- Microinjection of microcystin into hepatocytes was performed.
- Calmodulin antagonists and specific PP1 inhibitors (inhibitor-1, inhibitor-2) were used.
- Cellular morphology, surface protrusions, cell fragmentation, and DNA replication were assessed.
Main Results:
- Microcystin-LR and okadaic acid induced surface protrusions and cell fragmentation, mimicking apoptosis.
- Microinjected microcystin reproduced these effects at ~1 microM intracellular concentration.
- Okadaic acid, but not microcystin-LR, inhibited epidermal growth factor-stimulated DNA replication.
- Calmodulin antagonists counteracted okadaic acid's effect on DNA replication.
- PP1 inhibitors had no effect on DNA replication.
Conclusions:
- Moderate inhibition of protein phosphatase 2A (PP2A) by okadaic acid abolishes hepatocyte DNA replication.
- Microcystin-LR's lack of effect on DNA replication suggests PP2A is specifically involved in its regulation.
- PP1 inhibition does not impede DNA replication, indicating distinct roles for PP1 and PP2A in hepatocyte proliferation.
- Calmodulin signaling is implicated in the okadaic acid-induced inhibition of DNA replication.