p27(Kip1) Acts Downstream of N-Cadherin-mediated Cell Adhesion to Promote Myogenesis beyond Cell Cycle Regulation

It is widely acknowledged that cultured myoblasts can not differentiate at very low density. Here we analyzed the mechanism through which cell density influences myogenic differentiation in vitro. By comparing the behavior of C2C12 myoblasts at opposite cell densities, we found that, when cells are sparse, failure to undergo terminal differentiation is independent from cell cycle control and reflects the lack of p27(Kip1) and MyoD in proliferating myoblasts. We show that inhibition of p27(Kip1) expression impairs C2C12 cell differentiation at high density, while exogenous p27(Kip1) allows low-density cultured C2C12 cells to enter the differentiative program by regulating MyoD levels in undifferentiated myoblasts. We also demonstrate that the early induction of p27(Kip1) is a critical step of the N-cadherin-dependent signaling involved in myogenesis. Overall, our data support an active role of p27(Kip1) in the decision of myoblasts to commit to terminal differentiation, distinct from the regulation of cell proliferation, and identify a pathway that, reasonably, operates in vivo during myogenesis and might be part of the phenomenon known as “community effect”

Mean WBCs counts during the acute stage of infection.

<p>a) Mean WBCs ± SEM were plotted as a percentage of baseline counts in group A (low dose inoculum; only those animals that exhibited fever and/or positive by either RT-PCR or virus isolation were included e.g. 7 out of 12 calves) and group B (medium dose inoculum). WBC counts are shown for the acute phase of infection in group C (high dose inoculum). b) Mean WBCs ± SEM were plotted as percentages of baseline counts over the duration of the study, namely 60 dpi for group B (medium dose) and 71 dpi for group D (medium dose with DMS administered at time of inoculation).</p

Ct values for short target and negative strand RT-PCR.

<p>Ct values for short target and negative strand RT-PCR.</p

Summary of virological findings for increasing dose of Ho916 during acute infection and for treatment with and without DMS.

<p>Summary of virological findings for increasing dose of Ho916 during acute infection and for treatment with and without DMS.</p

Mean rectal temperatures during the acute stage of infection with low, medium, and high dose BVDV-1.

<p>Mean rectal temperatures ± SEM were plotted for calves inoculated with Ho916 a) Group A—low dose, 10^2.55 TCID₅₀, b) Group B—medium dose b—10^5.25 TCID₅₀ and c) Group C—high dose, 10^6.7 TCID₅₀, and for the sentinel animals in group A and B. Calves inoculated with a low dose were euthanised at 35 dpi. For the medium dose calves, only the acute phase of infection is shown, up to 38 dpi and 39 dpi for sentinel and infected calves respectively. In group C, the 7 week old calves were euthanised at 21 dpi, and for the 5 month old calves, only data from the acute phase of the infection (up to 21 dpi) is shown.</p