Joanna M. Watson

RhoA is required for monocyte tail retraction during transendothelial migration

Transendothelial migration of monocytes is the process by which monocytes leave the circulatory system and extravasate through the endothelial lining of the blood vessel wall and enter the underlying tissue. Transmigration requires coordination of alterations in cell shape and adhesive properties that are mediated by cytoskeletal dynamics. We have analyzed the function of RhoA in the cytoskeletal reorganizations that occur during transmigration. By loading monocytes with C3, an inhibitor of RhoA, we found that RhoA was required for transendothelial migration. We then examined individual steps of transmigration to explore the requirement for RhoA in extravasation. Our studies showed that RhoA was not required for monocyte attachment to the endothelium nor subsequent spreading of the monocyte on the endothelial surface. Time-lapse video microscopy analysis revealed that C3-loaded monocytes also had significant forward crawling movement on the endothelial monolayer and were able to invade between neighboring endothelial cells. However, RhoA was required to retract the tail of the migrating monocyte and complete diapedesis. We also demonstrate that p160ROCK, a serine/threonine kinase effector of RhoA, is both necessary and sufficient for RhoA-mediated tail retraction. Finally, we find that p160ROCK signaling negatively regulates integrin adhesions and that inhibition of RhoA results in an accumulation of β2 integrin in the unretracted tails.

Identification of the structural region of taxol that may be responsible for cytokine gene induction and cytotoxicity in human ovarian cancer cells

Purpose: Interleukin-8 (IL-8) is a pleiotropic chemokine with both chemoattractant and angiogenic properties. In addition to its cytotoxic effects on ovarian cancer cells, taxol can transcriptionally activate genes such as IL-8 that may play a role in tumorigenesis. Utilizing IL-8 as a prototypic marker of tumor-derived modulators of growth, we undertook a systematic study of taxol and 11 structurally modified taxol analogs to identify the region of the taxane skeleton responsible for IL-8 gene induction. Methods: The human ovarian cancer cell line OVCA-420 was exposed to taxol or taxol analogs. IL-8 gene induction was assessed by Northern blot analysis after 6 h and cytotoxicity after 72 h. Results: Changes in the southern hemisphere (C-1 to C-4) of the taxane skeleton had greater effects on IL-8 induction than changes in the northern hemisphere (C-7 to C-11). Some of the taxol analogs modified at positions C-1 and/or C-2 with increased hydrophobicity induced IL-8 expression more than threefold over that induced by taxol or taxotere and more than 20-fold over control cells. Cells that failed to induce IL-8 gene expression in response to taxol were only marginally responsive to the analogs unless first primed with IL-1β. Modifications to the northern hemisphere did not alter taxols effect on IL-8 expression in human cells, but did influence TNFα expression in murine macrophage cells, suggesting species and/or gene specificity. We found a direct correlation between IL-8 induction and cytotoxicity, in that analogs that dramatically upregulated IL-8 expression proved to be the most cytotoxic, inhibiting cell survival by >90%. Conclusion: Taken together our results demonstrate that changes in the southern hemisphere of the taxane skeleton influence both the gene induction and cytotoxic potential of taxol in human ovarian cancer cells.

IL-1β transcript stability in monocytes is linked to cytoskeletal reorganization and the availability of mRNA degradation factors

Monocyte extravasation initiates reorganization of the cytoskeleton (CSK) and adhesion‐dependent cytokine gene transcription. The actin CSK is thought to be crucial for compartmentalization and translation of mRNA, many of which contain AU‐rich (ARE) instability motifs in the 3′ untranslated region. We investigated regulation of adhesion‐induced IL‐1β expression by the monocyte CSK. In serum‐free adherent monocytes, the induced IL‐1β mRNA was stable and did not coextract with actin filaments. In contrast, in cells adherent in autologous serum, IL‐1β transcripts were unstable, coextracted with actin filaments and were associated with only transient activation of the mitogen‐activated protein kinases (MAPK). Under both conditions of adherence, the ARE‐binding protein AUF1/hnRNP D was readily extracted in the cytosolic fraction. Electro‐injection with AUF1/hnRNP D modified the actin CSK and, surprisingly, stabilized IL‐1β transcripts. These data suggest that the control of mRNA degradation is linked with changes in the CSK. Mitogen‐activated protein kinase activation or alterations in the availability of mRNA degradation factors may mediate these effects.