In the current study, we found that activation of PPARγ by Rosi, in primary and transformed MSCs, and in mouse liver cells, suppressed expression of IGF-I. Moreover, Rosi reduced expression of the IGF1R and IGFBP-4 in primary adult MSCs. Similarly, in mice and in postmenopausal women, we noted that Rosi treatment reduced circulating IGF-I concentrations by as much as 25%. The IGF-I circulatory changes occurred as early as 4 d after initiation of treatment, and were associated with reduced IGF-I transcripts in liver and peripheral fat. As such, these data raise a number of provocative questions, not only in respect to the effects of PPARγ activation on bone turnover, but also in regards to the mechanism of action of the PPARγ agonists.
This series of investigations started after we identified a major QTL for BMD and serum IGF-I between two inbred strains of mice, B6 and C3H (C3H/HeJ) (26
). This QTL was located on mouse chromosome 6 in the region of the Pparγ
). To test the biological effect of this QTL, we generated a congenic mouse (i.e.
6T) that carried C3H genes from this locus in a pure B6 background (43
). The resultant skeletal phenotype of 6T included low trabecular bone mass, increased marrow adiposity but enhanced insulin sensitivity, and low serum IGF-I (43
). Subsequent expression profiling of the liver and marrow revealed a pattern consistent with endogenous PPARγ
). To pursue this further, we then asked whether exogenous activation of PPARγ
could affect the IGF regulatory system in bone and liver.
activation could have a deleterious effect on bone through a number of possible mechanisms. First, Rosi
can directly suppress OB differentiation genes such as Runx2, Dlx5, and osterix, as nicely demonstrated in our microarray experiments () (9
). Second, work by our group and other investigators suggest there is an inverse relationship between the Wnt/β
-catenin signaling pathway and PPARγ
). Third, we now show that IGF-I is also down regulated in marrow stromal cells by Rosi,
as is the other important IGF ligand in bone, IGF-II. Changes in the bone IGF regulatory system in response to TZDs could affect the ultimate fate of pre-OBs, probably in combination with other key differentiation factors such as Runx2, osterix, and BMP4 (see ). For example, Zhang et al.
) demonstrated that targeted deletion of the IGF1R in OBs, using the Cre-loxP system with an osteocalcin promoter, markedly impaired two processes in late OB differentiation, matrix formation and mineralization. On the other hand, Jiang et al.
) demonstrated that targeted over-expression of IGF-I using a 3.6 Col1A1 promoter, resulted in greatly enhanced bone turnover, implying that IGF-I was important in pre-OB recruitment and may be a determinant of lineage allocation. Interestingly, aged B6 mice expressed significantly less IGF-I mRNA than adult B6 mice, and the suppressive effect of Rosi
on these cells was also dampened (see ). Because “old” MSCs have enhanced PPARγ
2 expression, it is interesting to speculate whether aging may affect IGF-I expression through this nuclear receptor activation (22
Another provocative aspect of these data is the acute suppression of IGF-I transcripts from the liver by 4 d of Rosi
administration. These findings were confirmed in vitro
by demonstrating that Rosi
reduced IGF-I mRNA by 50% in AML cells and in vivo
by demonstrating lower levels of circulating IGF-I without changes in the IGFBPs. It is conceivable that the suppressive effects of TZDs on serum IGF-I could not only contribute to the deleterious skeletal changes, but also to the antiproliferative and antineoplastic properties of these agents. In fact, we found several cell cycle genes in MSCs suppressed by activation of PPARγ
, some as early as 24 h after exposure. These findings are consistent with other reports which have noted the importance of cell cycle arrest in the antiproliferative response to PPARγ
). The mechanisms responsible for these changes are likely to be complex but may be partially mediated through suppression of IGF1R expression and its downstream signaling cascade (see ). However, more studies are needed to delineate the exact role of PPARγ
agonists in slowing cell proliferation and to define the proportion of change in cell proliferation that can be related to alterations in the IGF-I signaling network.
Several lines of evidence from our studies suggest that Rosi
directly suppresses liver IGF-I gene expression, rather than acting through the GH/IGF-I axis. First, we found reduced hepatic IGF-I transcripts in AML cells treated with Rosi
, independent of GH. Similarly, we found markedly suppressed IGF-I liver transcripts (i.e.
~60%) in B6 mice exposed to Rosi
for as little as 4 d, without changes in Stat5b, a major transcription factor induced by GH (50
). Second, serum IGFBP-3 did not change over the 16 wk of Rosi
treatment despite a drop of nearly 25% in IGF-I at 8 wk. And, there was no effect of Rosi
on IGFBP-3 expression in MSCs in vitro
. IGFBP-3 is induced by GH, hence any significant change in its secretion should be reflected in ambient IGFBP-3 concentrations or tissue expression. Our results are similar to those of Bell et al.
), who found that women with polycystic ovary syndrome treated with Rosi
had significant suppression of serum IGF-I but not IGFBP-3. Third, we found in the microarray experiments that the GHR was actually up-regulated by exposure of U-33/γ
2 cells to Rosi
. As such, these changes may have clinical significance. A drop in circulating IGF-I levels could negatively impact the skeleton, particularly in younger individuals who may be insulin resistant yet are still acquiring peak BMD (41
). On the other hand, low serum IGF-I concentrations could limit cell proliferation and enhance apoptosis in specific target tissues. It will be interesting to see whether changes in serum IGF-I can predict the effects of Rosi
on a host of proliferative disorders.
There are several limitations to this study. First, we only examined one inbred strain, B6, to determine the effects of Rosi
on IGF-I. It is conceivable that there are strain-related differences in the IGF-I response to activation of PPARγ
, particularly in light of our previous genetic studies in B6 and C3H mice (43
). Another limitation is that we chose MSCs rather than OBs to study the effects of Rosi.
Hence, we do not know whether TZDs can impact the differentiative function of mature OBs, particularly matrix production and mineralization. In addition, we have not explored the mechanism responsible for enhanced IGF-I expression in MSCs that do not have endogenous PPARγ
2 activity (i.e.
U-33/c cells), although unlike U-33/γ
2 cells, these cells are biologically unique in that they cannot differentiate into adipocytes. Also, there is emerging evidence that not all TZDs work on adipocytes and OBs in the same manner (21
). Studies with other ligand agonists should provide further insight as to whether the IGF suppressive effects are a class property or unique to Rosi
. A limitation to the human study was the small number of subjects and the duration of the trial was short (i.e.
16 wk). It is uncertain whether suppression of IGF-I is persistent with chronic Rosi
therapy, or whether GH secretion is enhanced by the drop in IGF-I, resulting in restoration of serum IGF-I levels. That scenario is plausible, particularly because we showed that Rosi
enhances expression of the GHR in MSCs at 24 and 72 h. However, suppression of the IGF1R by Rosi
means that even with restoration of circulating levels, the bioactivity of IGF-I could be compromised.
In conclusion, we established that activation of PPARγ suppresses elements of the IGF regulatory system in pre-OBs and in liver. This effect is evident within 72 h and occurs at the transcriptional and/or posttranscriptional levels, affecting both IGF-I mRNA and protein production. Similar changes occur in vivo as early as 96 h after exposure. The downstream consequences of reduced expression of IGF-I, its receptor, and several signaling factors, may contribute to the unique properties of the TZDs. Further studies should provide greater insight into the role IGF-I plays in mediating the antiosteoblastic actions of agents such as Rosi.