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Induced expression of Rnd3 is associated with transformation of polarized epithelial cells by the
S H Hansen1, M M Zegers, M Woodrow
1Cancer Research Institute and Department of Cellular and Molecular Pharmacology, University of California San Francisco Cancer Center, San Francisco, California 94143, USA. hansen@bbri.org
Abstract:
Madin-Darby canine kidney (MDCK) epithelial cells transformed by oncogenic Ras and Raf exhibit cell multilayering and alterations in the actin cytoskeleton. The changes in the actin cytoskeleton comprise a loss of actin stress fibers and enhanced cortical actin. Using MDCK cells expressing a conditionally active form of Raf, we have explored the molecular mechanisms that underlie these observations. Raf activation elicited a robust increase in Rac1 activity consistent with the observed increase in cortical actin. Loss of actin stress fibers is indicative of attenuated Rho function, but no change in Rho-GTP levels was detected following Raf activation. However, the loss of actin stress fibers in Raf-transformed cells was preceded by the induced expression of Rnd3, an endogenous inhibitor of Rho protein function. Expression of Rnd3 alone at levels equivalent to those observed following Raf transformation led to a substantial loss of actin stress fibers. Moreover, cells expressing activated RhoA failed to multilayer in response to Raf. Pharmacological inhibition of MEK activation prevented all of the biological and biochemical changes described above. Consequently, the data are consistent with a role for induced Rnd3 expression downstream of the Raf-MEK-extracellular signal-regulated kinase pathway in epithelial oncogenesis.
Insights
Oncogenic Ras and Raf transformation of kidney cells induces multilayering by altering actin cytoskeleton dynamics. This involves increased Rac1 activity and Rnd3 expression, inhibiting Rho, leading to stress fiber loss and cell changes.
Area of Science:
- Cell Biology
- Molecular Biology
- Oncogenesis
Background:
- Oncogenic Ras and Raf transformations in Madin-Darby canine kidney (MDCK) cells cause cell multilayering and actin cytoskeleton alterations.
- These changes include loss of actin stress fibers and increased cortical actin.
Purpose of the Study:
- To investigate the molecular mechanisms behind Ras/Raf-induced changes in MDCK cell actin cytoskeleton and multilayering.
- To elucidate the role of Rho family GTPases and their regulators in this process.
Main Methods:
- Utilized MDCK cells expressing a conditionally active form of Raf.
- Assessed Rac1 and Rho GTPase activity.
- Measured Rnd3 expression levels.
- Examined actin stress fiber organization and cortical actin.
- Investigated the effect of activated RhoA and MEK inhibition.
Main Results:
- Raf activation increased Rac1 activity and cortical actin.
- Raf activation induced Rnd3 expression, which inhibited Rho function and caused stress fiber loss.
- Activated RhoA expression prevented Raf-induced multilayering.
- MEK inhibition blocked all observed biological and biochemical changes.
Conclusions:
- Induced Rnd3 expression downstream of the Raf-MEK-ERK pathway plays a critical role in Ras/Raf-mediated epithelial oncogenesis.
- The pathway regulates actin cytoskeleton dynamics, influencing cell morphology and behavior.