Osteoclast differentiation is impaired in the absence of inhibitor of kappa B kinase alpha.

Signaling through the receptor activator of nuclear factor kappa B (RANK) is required for both osteoclast differentiation and mammary gland development, yet the extent to which RANK utilizes similar signaling pathways in these tissues remains unclear. Mice expressing a kinase-inactive form of the inhibitor of kappa B kinase alpha (IKK alpha) have mammary gland defects similar to those of RANK-null mice yet have apparently normal osteoclast function. Because mice that completely lack IKK alpha have severe skin and skeletal defects that are not associated with IKK alpha-kinase activity, we wished to directly examine osteoclastogenesis in IKK alpha(-/-) mice. We found that unlike RANK-null mice, which completely lack osteoclasts, IKK alpha(-/-) mice did possess normal numbers of TRAP(+) osteoclasts. However, only 32% of these cells were multinucleated compared with 57% in wild-type littermates. A more profound defect in osteoclastogenesis was observed in vitro using IKK alpha(-/-) hematopoietic cells treated with colony-stimulating factor 1 and RANK ligand (RANKL), as the cells failed to form large, multinucleated osteoclasts. Additionally, overall RANKL-induced global gene expression was significantly blunted in IKK alpha(-/-) cells, including osteoclast-specific genes such as TRAP, MMP-9, and c-Src. IKK alpha was not required for RANKL-mediated I kappa B alpha degradation or phosphorylation of mitogen-activated protein kinases but was required for RANKL-induced p100 processing. Treatment of IKK alpha(-/-) cells with tumor necrosis factor alpha (TNF alpha) in combination with RANKL led to partial rescue of osteoclastogenesis despite a lack of p100 processing. However, the ability of TNF alpha alone or in combination with transforming growth factor beta to induce osteoclast differentiation was dependent on IKK alpha, suggesting that synergy between RANKL and TNFalpha can overcome p100 processing defects in IKK alpha(-/-) cells.

Hepatocyte-specific inhibition of NF-kappaB leads to apoptosis after TNF treatment, but not after partial hepatectomy.

One of the earliest TNF-dependent events to occur during liver regeneration is the activation of the transcription factor NF-kappaB through TNF receptor type 1. NF-kappaB activation in the liver can have both antiapoptotic and proliferative effects, but it is unclear which liver cell types, hepatocytes or nonparenchymal cells (NPCs), contribute to these effects. To specifically evaluate the role of hepatocyte NF-kappaB, we created GLVP/DeltaN-IkappaB(alpha) transgenic mice, in which expression of a deletion mutant of IkappaB(alpha) (DeltaN-IkappaB(alpha)) was induced in hepatocytes after injection of mifepristone. In control mice, injection of 25 microg/kg TNF caused NF-kappaB nuclear translocation in virtually all hepatocytes by 30 minutes and no detectable apoptosis, while in mice expressing DeltaN-IkappaB(alpha), NF-kappaB nuclear translocation was blocked in 45% of hepatocytes, leading to apoptosis 4 hours after TNF injection. In contrast, expression of DeltaN-IkappaBalpha in hepatocytes during the first several hours after partial hepatectomy did not lead to apoptosis or decreased proliferation. As NF-kappaB activation was not inhibited in liver NPCs, it is likely that these cells are responsible for mediating the proliferative and antiapoptotic effects of NF-kappaB during liver regeneration.

Bcl-2 expression delays hepatocyte cell cycle progression during liver regeneration.

Bcl-2 is the prototype of a family of genes that prevent apoptosis. However, several reports indicate that Bcl-2 may also act as a cell cycle modulator. In several human tumors, Bcl-2 expression correlates with a more favorable prognosis and lower tumor proliferative activity. We have shown that Bcl-2 expression delays liver tumor development in transgenic mice even when the gene is turned on shortly before the time of tumor development. We hypothesized that Bcl-2 may delay liver tumorigenesis by interfering with hepatocyte proliferation. To test whether Bcl-2 expression may act on hepatocyte replication we studied liver regeneration in Bcl-2 transgenic mice and wild-type littermates. DNA replication was delayed by approximately 8 h in Bcl-2 transgenic mice compared to the timing of the response in wild-type littermates. Cyclin D expression showed no alterations in the regenerating liver of Bcl-2 transgenic mice. In contrast, there was a delay in the expression of p107, cyclin E and in the activity of cyclin E/cdk 2 activity. These results show that Bcl-2 expression delays cell cycle progression in hepatocytes and suggests that it acts at a step involving cyclin E and p107.