The hindlimb suspension model is used as an animal model to investigate the mechanisms of bone loss caused by unloading from disuse or space flight. With this model, we and others have demonstrated that skeletal unloading causes suppression of osteoblast numbers, proliferation, and bone formation. We have demonstrated that glucocorticoids have a minimal effect on osteoblast function as we have found plasma corticosterone levels during hindlimb unloading are the same as those found in control animals, and adrenalectomy does not alter the unloading effect on bones.(34)
The bone marrow stromal cells isolated from unloaded bones express a higher level of the adipocyte differentiation marker, PPARγ2, suggesting a lower osteogenic potential. However, the level of Runx2 expression in these adherent cells is not decreased, suggesting there is a pool of available osteoprogenitor cells, but they are resistant to bone formation signals. The cross-talk and stimulation of osteoclasts may also be impaired as there is lower propensity to form osteoclasts; the number of TRAP+ osteoclasts is 63% of controls, p < 0.05 (not shown).
Thus, we considered the possibility that the response of osteoblasts and osteoblast precursors to receptor tyrosine kinase growth factors would be impaired by skeletal unloading and not to non-receptor tyrosine kinase factors like TGFβ, that have been shown to blunt unloading induced bone loss.(29)
In this study, we found that this expectation was only realized, in that not all receptor tyrosine kinase growth factors were impacted. In previous studies(5,6,35,36)
we have shown that resistance to IGF-I plays an important role in the impaired osteoblast function and decrease in bone formation during skeletal unloading. The IGF-I resistance in bones caused by skeletal unloading persists in the BMOp cells isolated from unloaded bones. Our present results demonstrate that the resistance to IGF-I caused by skeletal unloading is not shared by PDGF. PDGF, unlike IGF-I, signaling was fully intact and capable of stimulating BMOp cells from unloaded bones.
Unloading induced resistance of IGF-I is characterized by a failure of IGF-I to stimulate cognate receptor phosphorylation and to activate proliferative and anti-apoptotic signaling cascades. We show that this is not a function of decreased receptor quantity or impaired IGF-I receptor affinity. Unloading does not induce changes in IGF binding proteins as ligand binding affinity curves are unchanged with des-IGF-I, an analogue with minimal binding protein affinity. PDGF signaling, on the other hand, is unaffected by skeletal unloading as demonstrated by completely intact PDGF receptor and downstream signaling phosphorylation in response to PDGF.
The difference in integrity of the PDGF and IGF-I signaling cascades in response to skeletal unloading is due to selective integrin receptor regulation of IGF-I but not PDGF signaling in BMOp cells. Previously, we demonstrated that αVβ3 expression in BMOp cells was reduced by skeletal unloading, and echistatin, a disintegrin selective for αVβ3 or α5β1, blocked IGF-IR phosphorylation.(6)
Echistatin causes an IGF-I resistance phenomenon similar to that induced by unloading, but it fails to block ligand induced PDGF receptor activation. Skeletal unloading induces a dramatic reduction of numerous integrin subunits, and despite this reduction skeletal unloading fails to block PDGF signaling.
The marked reduction of integrin subunits in unloaded BMOp cells provides a mechanism of the “memory” BMOp cells have of their previous loaded state. BMOp cells demonstrate unloading induced resistance to IGF-I both in vivo and in vitro. The memory of the unloaded state is a transient phenomenon in vitro. In recently isolated unloaded BMOp cells integrin expression is reduced, which causes IGF-I resistance and contributes to the reduction in cell colonies formed.(5)
However, as these cultures grow these cells recover integrin expression, as well as IGF-I signaling and receptor phosphorylation. This recovery of unloaded cells is contemporary with BMOp differentiation and maturation, with normal alkaline phosphatase and mineralization activity per cell noted.(5)
Integrin subunits are markers of functional osteoblasts.
Integrin regulation of IGF-I signaling has been described in various tissues. Impaired IGF-I receptor activation by disruption of ligand occupancy of integrins with echistatin,(6,37)
and ligand independent activation of IGF-I receptor by mechanical stimulation of osteosarcoma cells with fluid flow(37)
demonstrates a role of integrins in IGF-I signaling. However, a specific integrin regulator of IGF-I signaling in osteoblasts and osteoblast precursors is unknown. Studies from Clemmons and co-workers(22,23)
have detailed a proposed mechanism of interaction between the integrin and IGF-I signaling pathways. In porcine vascular smooth muscle cells, integrin β3 phosphorylation recruits the phosphatase SHP-2 to the membrane and enables the transfer of SHP-2 to SHPS-1 away from the IGF-I receptor, enhancing IGF-I receptor phosphorylation and signaling. Blocking integrin β3 activation prevents SHP-2 sequestering and terminates IGF-I signaling.
The SHP-2 oriented mechanism incompletely describes the nature of integrin regulation of the IGF-I receptor in osteoblasts. We have shown that unloading induced impairment of IGF-I receptor activation is not recovered by pretreatment with the phosphatase inhibitor orthovanadate,(6)
thus IGF-I resistance is not a function of increased phosphatase activity but rather a loss of kinase activity. Mechanical stimulation of osteosarcoma cells in a serum-free environment phosphorylated the IGF-I receptor, which was blocked by the disintegrin, echistatin.(37)
These observations suggest activity of an integrin mediated non-receptor kinase is required for IGF-I receptor activation. Recently, IGF-I has been shown to directly bind integrin β3 and to stimulate a complex formation of the IGF-I receptor and αVβ3 integrin. IGF-1 mutated to disrupt integrin β3 binding only retained IGF-I receptor binding but failed to phosphorylate the IGF-I receptor, activate downstream AKT and ERK signaling pathways, stimulate proliferation, and stimulate integrin/IGF-I receptor complex formation.(38)
This complex would allow cross talk between the two signaling pathways and recruitment of non-receptor kinases to directly phosphorylate the IGF-I receptor. Focal adhesion kinases, components of the integrin signaling cascade, are required for growth factor response, regulating response with intrinsic kinase activity or providing binding sites for other non-receptor kinases.(39,40)
Specifically, FAK and PYK2 have been shown to directly interact with the insulin and IGF-I receptors.(41,42)
We used siRNA knockdown to target integrin β1 and β3 specifically to determine whether either integrin subunit regulates IGF-I signaling in BMOp cells. We did not assess the effect on PDGF signaling as unloading induced reductions in integrin expression did not affect PDGFR activation. Integrin β1 was included as a candidate subunit because it is predominately expressed in bone and, like β3, has been shown to modulate IGF-I signaling(43,44)
in other cell types. Our results show that a reduction in β3 expression significantly reduced IGF-I receptor phosphorylation whereas a reduction in β1 expression had a less consistent effect. This may indicate that β3 is more critical for IGF-I signaling than β1 or that because of the high level of expression of β1 in these cells sufficient β1 levels remain after siβ1 treatment to maintain some level of IGF-I responsiveness. In either case the siRNA knockdown studies demonstrate that integrin β3, and possibly β1, function as regulators of the IGF-I receptor in rat BMOp cells that may be responsive to mechanical stimulation. The siRNA knockdown model was not equivalent to the skeletal unloading model as it was not sufficient to assess the effect of targeted integrin knockdown on signaling downstream of IGF-IR. Although IGF-IR phosphorylation was specifically manipulated by siRNA treatment, basal pERK levels were uniformly increased in response to siRNA treatment, and this lowered the sensitivity to detects changes in response to IGF-I exposure.
In conclusion, skeletal unloading causes bone loss that is associated with impaired IGF-I but not PDGF signaling. Our results indicate that the mechanism by which skeletal unloading leads to IGF-I resistance has little impact on the proliferative response to PDGF. IGF-I signaling in osteoblast precursors is more sensitive to the unloading induced reductions in integrins, especially β3, than PDGF. The integrity of PDGF signaling can be utilized as PDGF is known to stimulate osteoblast proliferation and differentiation.(45)
Localized use of PDGF therapy to repair periodontal defects is a FDA approved indication, and systemic use in an osteoporotic animal model demonstrated increased osteoblast proliferation, bone formation, and BMD.(46)
Potentially, PDGF may be an option to treat bone loss or prevent disuse osteoporosis in conditions of skeletal unloading like prolonged bedrest, space flight, or neurologic injury.