Increased integrin expression in differentiated OC-2 cells
To confirm that OC-2 cells grown at 39°C for 2 weeks had differentiated, myosin VI and myosin VIIa levels were examined by Western blot analysis. As has been shown previously [14
], differentiated OC-2 cells increased their expression of both myosin VI and VIIa (Figure ) (P
< 0.01) with respect to undifferentiated cells.
Figure 1 Integrin expression profiles are altered after OC-2 cell differentiation. (A) Western blot analysis of myosin VI and myosin VIIa levels in OC-2 cells grown at 33°C and 39°C. Both myosin VI and VIIa levels were increased significantly in (more ...)
FACS and Western blot analysis were utilized to quantify the relative changes of expression of the integrin subunits during the differentiation of OC-2 cells. OC-2 cells were screened for integrin expression and the integrin subunits α6, αv, β1 and β3 were detected (Figure ) but not α1-, α2-, α5- and β4-integrin subunits (data not shown). The expression of α6-integrin subunit was not significantly different between undifferentiated and differentiated OC-2 cells. In contrast, αv- and β1-integrin surface levels were increased in differentiated OC-2 cells when compared with undifferentiated OC-2 cells (P < 0.01) (Figure ). No expression of β3-integrin was observed in undifferentiated OC-2 cells but this receptor was expressed after the OC-2 cells had undergone differentiation (P < 0.01). Similar results were obtained when total protein levels of these integrin subunits were examined by Western blot analysis (data not shown). To verify our in vitro findings, RT-PCR for the integrin subunits α6, αv, β1, β3 and β4 (as a control) was performed on RNA isolated from cochlear tissue of adult mice (Figure ). All integrin subunits examined were expressed in mouse cochlear tissue, to varying levels, apart from β4-integrin, which also was not expressed on OC-2 cells.
Figure 2 Integrin expression profiles in the Organ of Corti. RT-PCR of RNA isolated from organ of Corti of adult mice. Bands at the predicted molecular weight are visible for integrin β1, β3, αv, α6 and β-actin but not for (more ...)
Time course of integrin expression profiles during differentiation
Having shown that differentiated OC-2 cells had increased expression of αv, β1 and β3-integrin, we analysed the pattern of integrin expression over the 14 day differentiation period. OC-2 cells, cultured at 33°C, were transferred to 39°C and integrin expression was examined every 2 days for 14 days, when the cells had fully differentiated. Unsurprisingly, α6-integrin levels did not alter during the 14 day differentiation period (Figure ). αv-integrin expression levels remained similar to levels seen at 33°C cells for the first 4 days during differentiation. By day 6 of differentiation, αv-integrin expression levels increased to the levels observed in fully differentiated OC-2 cells (Figure ), and remained so until OC-2 cells were fully differentiated (day 14). However, we did observe that at day 10 of differentiation, the level of αv-integrin expression was increased to a level above that found in fully differentiated OC-2 cells.
Figure 3 αv-integrin expression profiles change during differentiation. Expression of (A) α6-integrin and (B) αv-integrin were examined by FACS at days 2, 6, 10 and 14 during the differentiation process. The differentiating cells ('dif' (more ...)
β1-integrin expression increased from day 2 of differentiation when compared with undifferentiated OC-2 cells. β1-integrin levels gradually increased until day 10, at which time β1-integrin surface expression was comparable to levels observed in fully differentiated OC-2 cells (Figure ). It was noted that throughout the early stages of differentiation (day 2-8), a small population of differentiating OC-2 cells expressed β1-integrin at a level comparable with fully differentiated OC-2 cells. No surface expression of β3-integrin was seen during the first 2 days of the differentiation process, but by day 4, expression of this integrin increased with time, reaching the level of expression observed in differentiated OC-2 cells by day 12 (Figure ). Western blot analysis of all the described integrin subunits showed similar patterns of integrin expression during the differentiation process, while the increase in myosin VIIa and myosin VI that occurs with differentiation (Figure ) started being evident from day 8 and day 12 respectively (data not shown).
Figure 4 β1 and β3-integrin expression profiles change during differentiation. Surface expression of (A) β1-integrin and (B) β3-integrin was examined by FACS at days 2, 6, 10 and 14 of the differentiation process The differentiating (more ...)
Functional correlation between integrin expression and myosin VIIa levels
Having shown that changes in the integrin expression profiles during differentiation coincided with changes in myosin VI and myosin VIIa levels, we wished to confirm a functional role for these integrins in this process. Fully differentiated OC-2 cells were transfected with siRNA targeted specifically to either αv-, α6-, β1- or β3-integrin subunits or a scrambled control siRNA, followed by analysis of myosin VIIa levels. We optimised the time of maximum effect of siRNA on the respective protein surface expression (24 hrs) and confirmed siRNA treatment had no effect on cell proliferation and was non-toxic to the cells (data not shown).
Western blot analysis showed that siRNA inhibition of all integrin subunits significantly reduced the level of expression of myosin VIIa in comparison with fully differentiated OC-2 cells, to levels similar to those observed in undifferentiated OC-2 cells (P < 0.01). Myosin VIIa levels were decreased most effectively in the presence of αv and β3-integrin siRNA (Figure ). To investigate the specificity of action of the integrin subunits we have measured the effect of some of the integrin siRNA on the expression of other integrin subunits. All integrin siRNAs, but not scrambled siRNAs significantly reduced the surface expression of their respective proteins and in some cases also other integrins. For example (Figure ) β3-siRNA caused a significant reduction of surface expression of β3, αv, β1 but not α6. Other examples of non-significant effect of siRNAs were α6-siRNAand αv-siRNA on β1 expression (data not shown). These data indicate that the effect of the siRNAs is specific but there is interdependency in the surface expression of different integrin subunits.
Figure 5 β3-integrin is a marker for OC-2 cell differentiation. (A) Western blot analysis of myosin VIIa expression in 39°C OC-2 cells untreated or after treatment with either scrambled (Scr), β1-, α6-, αv- and β3-integrin (more ...)
Figure 6 Effect of β3-integrin-siRNA treatment on integrin surface expression. Differentiated OC2 cells were treated with β3-siRNA and assayed by FACS for expression of surface integrins. The data is compared to integrin expression levels in normal (more ...)
Having shown that β3-integrin diplayed the most dramatic change in integrin expression during madifferentiation of the OC2 cells and that knockdown of this integrin was the most effective at reducing the expression of myosin VIIa, we over expressed β3-integrin in undifferentiated OC-2 cells to generate a phenotype similar to fully differentiated OC-2 cells. Retroviral transduction of undifferentiated OC-2 cells with human β3-integrin was confirmed by FACS analysis (Figure ) and showed surface expression levels similar to those observed in fully differentiated OC-2 cells. This over expression of β3-integrin, in turn, increased the expression of myosin VIIa to levels similar to those in fully differentiated OC-2 cells. Indeed, myosin VIIa levels were increased significantly in transduced cells when compared with undifferentiated OC-2 cells. The effect of over expression of human β3-integrin on other integrin expression patterns was examined by FACS analysis. α6-integrin surface levels did not change in response to increased β3-integrin, whereas αv-integrin, and, to a lesser extent, β1-integrin showed increased surface expression after transduction with human β3-integrin (data not shown).