The PI3K pathway has been shown to affect numerous cellular processes in a tissue-specific fashion; for example, it is required for survival in different cell types such as cardiomyocytes , cellular differentiation in the case of osteoclasts and keratinocytes [35, 36], and proliferation and differentiation of osteoblasts . It also stimulates differentiation of CD4+ T-cells  and development and proliferation of B cells [38, 39]. We hypothesized that the PI3K pathway has similar effects in the growth plate, promoting endochondral bone growth by increasing proliferation and differentiation of chondrocytes and by suppressing apoptosis.
We found that inhibition of PI3K with LY294002 results in decreased differentiation, in both primary chondrocytes (micromass cultures) and organ cultures. Markers of both early chondrocyte differentiation such as collagen II and glycosaminoglycans and of late hypertrophic differentiation such as collagen X, p57, Alkaline phosphatase activity and calcium content (Alizarin red S stain) were decreased upon PI3K inhibition. These data suggest that the PI3K pathway is required for normal chondrocyte differentiation. In the organ culture system, we have shown that the PI3K pathway is required for maximal bone growth, since by inhibiting the pathway we obtained 55% reduction in bone growth, due to a proportionate shortening of both growth plates.
The major phenotype of the LY294002 treated tibiae is represented by a 45% reduction in the length of the hypertrophic zone, providing further evidence that the PI3K pathway is required for hypertrophic differentiation. The observed reduction in the area staining for collagen X and p57 in LY294002 treated tibiae is in agreement with reduced hypertrophy. In addition we have seen a 20% reduction of the length of the proliferative area of the growth plate, in LY294002 treated tibiae. In the organ culture system it seems that the onset of proliferation is delayed, since the resting zone represents a higher percentage of the growth plate in the LY294002 treated bones compared to control. The ratio of BrdU labeled cells within the proliferative zone of the growth plate does not appear to be different between LY294002 and control cultures, suggesting that PI3K inhibition results in delayed cell cycle entry, but does not affect the rate of cell cycle progression once cells have entered to proliferative zone. Our data also show increased apoptosis in organ cultures treated with LY294002. Apoptosis was only detected in the hypertrophic and mineralized zones, suggesting that the PI3K pathway is required for hypertrophic chondrocyte survival.
PI3K signaling transduces signals from many growth factors and other extracellular cues, but it is not known which of them utilizes the pathway for anabolic effects on endochondral bones. Potential candidates are IGFs, however, our data suggest, somewhat unexpectedly that IGF1 stimulates organ culture growth in the presence of LY294002 to a similar degree as in control cultures. IGF1 treatment causes an increase in the length of hypertrophic zone , and this increase is not completely blocked by the PI3K inhibitor. This suggests that the PI3K pathway is not the only and potentially not the major pathway required for IGF1-induced bone growth and -hypertrophic differentiation in our organ culture system.
One potential problem that could partially explain the lack of growth reduction in the IGF1 + LY294002 treatment is that IGF1 possibly increased Akt phosphorylation to a level that is no longer completely inhibited by 10 μM LY294002. The mechanisms for IGF1 and CNP regulation of the PI3K pathway in growth plate chondrocytes are not the focus of this manuscript, but we plan to investigate the implications of these two growth factors in more depth in future studies. It will be important to see the levels of phosphorylated Akt in all treatment combinations, by performing immunohistochemistry and Western blotting with protein isolated directly from the tibiae treated with all treatment combinations (control, IGF1, LY294002 and LY294002 + IGF1 or control, CNP, LY294002, and CNP +LY294002). In addition, future measurements of growth plate zones under all conditions might provide an explanation for the sustained anabolic effects of IGF1even in the presence of LY294002. Our results have shown that the PI3K pathway is mostly involved in hypertrophic chondrocyte differentiation, and also that the two ligands IGF1 and CNP increase the length of hypertrophic zone (Figures 6C and 7B). There were no obvious effects on the other zones, but performing growth plate zone measurements and molecular analyses (e.g. BrdU labeling) might bring additional information in the future.
It will also be of interest to determine which other pathways mediate anabolic activities of IGFs in cartilage; the other major signaling pathway implicated in IGF signaling in other cells, the MEK-ERK (mitogen-activated protein kinase kinase/extracellular regulated kinase) cascade, has been shown to suppress endochondral bone growth [33, 40–42] and is therefore an unlikely candidate for this role.
Surprisingly, C-type natriuretic peptide (CNP), which is not a known PI3K activator, was found to partially require PI3K activity to stimulate bone growth. The CNP-induced growth of cultured tibiae was blocked by the PI3K inhibitor. One interesting finding was that the effect of CNP on hypertrophy – a significant increase in the hypertrophic zone length  – was inhibited by LY294002. These data identify CNP as one signal requiring PI3K activity in cartilage, but there are other potential candidates for regulation of the PI3K pathway in endochondral bone growth, such as PTHrP (Parathyroid hormone-related protein) and integrin ligands. Studies are under way in our laboratory to identify physiological activators of PI3K signaling in cartilage.
The molecular mechanisms mediating the effects of PI3K signaling in endochondral bone growth remain to be identified. We show that Akt proteins are phosphorylated under control conditions, and that this activation is reduced under PI3K inhibition, resulting in reduced bone growth, in agreement with reduced growth in Akt1-deficient mice as well as mice deficient in multiple Akt genes[43, 44]. The PI3K/Akt pathway was shown to be involved in Runx2 (runt related transcription factor 2) -dependent osteoblast and chondrocyte differentiation in 2 cell lines, MC3T3-E1 and ATCDC5, respectively . Therefore it represents a candidate for the PI3K involvement in chondrocyte hypertrophy. Further investigations of the PI3K/Akt mechanisms of chondrocyte differentiation are necessary in order to find the direct targets of this signaling pathway.