Understanding the molecular mechanisms involved in adipocyte differentiation is of great importance because it may lead to new ways of treating obesity and, in particular, its associated metabolic complications. Hh signaling and IFN-γ already have an established role in inhibiting adipogenesis (21
Here we demonstrate for the first time that inhibition of adipocyte differentiation by Hh signaling can be balanced by the antagonistic actions of IFN-γ. The effect was observed in 3T3-L1 adipocytes () as well as in three different primary culture systems—mouse embryonic fibroblasts (), primary mouse preadipocytes (), and primary human preadipocytes (Supplementary Fig. 4I and J
). These data, as well as recapitulation of the phenotype using the endogenous Shh
ligand, rule out cell-line or chemical artifacts. The difficulty associated with recapitulating these studies in mature primary adipocytes is caused by the direct inhibition of Hh signaling by both IBMX and Dex, required constituents of primary adipocyte differentiation cocktails.
Cross-talk between the Hh signaling pathway and IFN-γ has already been shown to influence cell differentiation and proliferation in specific neuronal settings (31
). IFN-γ indirectly regulates proliferation of neuronal precursor cells through mechanisms involving elevated expression of Shh
and its major target genes, Gli1
). Discordant upregulation of Shh
by IFN-γ, while suppressing its target Gli1
, was observed in studies on the differentiation of neuronal stem cells (32
). Interestingly, our studies demonstrate that IFN-γ–mediated inhibition of Hh target genes (Gli1
, and Nr2f2/Coup-Tf2
) in 3T3-L1 cells promotes adipocyte differentiation. IFN-γ not only rescued adipocyte differentiation but also decreased Coup-Tf2 and Gata2 levels, further supporting an antiadipogenic role of both genes (32
). Thus, it appears that IFN-γ signaling can have very context-dependent effects on Hh signaling.
After binding of IFN-γ to its receptor, Jak1 and Jak2 are activated and regulate downstream phosphorylation of Stat1 (46
). Although Stat1 is required for many IFN-γ–dependent actions, evidence shows that in the absence of Stat1, IFN-γ can still regulate the expression of some genes (46
). Because IFN-γ–mediated inhibition of Hh signaling did not occur in Stat1−/−
MEFs, we conclude that this cross-talk depends on Stat1. Interestingly, blocking constitutive Jak-Stat activation by a pan-Jak inhibitor resulted in greatly increased Gli1 luciferase activities in SAG-treated Shh-LIGHT2 cells, even in the absence of IFN-γ. This suggests that basal Jak activity exerts an inhibitory tone on Hh pathway activation.
Because the concentration of IFN-γ is elevated in obesity and the Hh signaling pathway was less active in obese compared with lean mice, we hypothesized that IFN-γ might inhibit Hh signaling in vivo. Interestingly, when examining adipose tissue fractions for differences in Gli1
expression in the lean and obese state, we found low Gli1
expression in mature adipocytes. Levels of mRNA in dSVF were >20-fold and in macrophages >40-fold higher than in the adipocyte fraction. This is in accordance with previous data that show decreased expression of positively acting Hh components (Smo and the Glis) during adipogenesis (29
). To determine whether reduced expression of Hh target genes in white fat of obese animals depends on higher IFN-γ concentrations in obesity, we treated obese mice with a neutralizing antibody against IFN-γ. Our results demonstrate that obesity-associated repression of Hh-target genes like Gli1
in the dSVF was partially or completely relieved by inhibiting IFN-γ elicited Jak/Stat signaling. This suggests that IFN-γ opposes inhibition of Hh signaling in obese adipose tissue in vivo.
Increases in fat mass can involve both hypertrophy and hyperplasia of adipocytes. Obesity in adults is typically associated with adipocyte hypertrophy, and an additional adipocyte hyperplasia occurs in morbidly obese humans and rodents (48
). Adiposity-induced cellular and tissue hypoxia is an important contributor to adipose tissue immunopathies and adipocyte and adipose tissue dysfunction (2
). Data suggest that the initial hypertrophic response of adipocytes promotes necrotic-like death, after which the lipid storage capacity of adipose tissue can be efficiently maintained or increased only by adipocyte hyperplasia. Interestingly, current concepts support that early adipose tissue expandability prevents metabolic consequences like insulin resistance, even in morbid obesity (9
Some have proposed that the generation of new adipocytes may be balanced by adipocyte death, with the constant and tightly regulated total number set by early adulthood (50
). Our findings suggest a model where the inhibition of Hh signaling by IFN-γ in obesity contributes to the development of new adipocytes (). At steady-state in lean individuals, basal Hh-pathway activity prevents the formation of new adipocytes. T-cell infiltration, as an early event in obesity development, leads to elevated IFN-γ production in adipose tissue and inhibition of Hh-signaling, a scenario that is permissive for the recruitment and development of new adipocyte populations. Such an early inhibition of Hh signaling may be a prerequisite for compensatory increases in total adipocyte numbers, although that is speculation.
FIG. 6. Model shows a novel cross-talk mechanism between IFN-γ and Hh signaling maintains adipogenesis in adipose tissue. Activation of Hh signaling blocks white adipocyte differentiation. T cells infiltrating white adipose tissue early during the development (more ...)
Our observation that the main Hh target gene GLI1
is downregulated in intraperitoneal adipose tissue from metabolically healthy IS obese subjects supports the concept that early inactivation of Hh signaling and subsequent release of the intracellular progenitor pool to expansion might alleviate obesity associated insulin resistance. Clearly though, the system is more complex. Indeed, robust opposing signals for T-cell infiltration (upregulated) and Hh signaling (downregulated) in the adipose tissue of MZ twins as well as in Pima Indian cohorts support the idea that inhibition of Hh signaling alone is insufficient to block pathologies associated with obesity and chronic inflammation (Supplementary Fig. 9
). It is almost certain that additional positive expansion signals are required for proper expansion. A deeper understanding of these concepts will be aided by long-term genetic dissections in vivo, using for instance inducible adipose tissue specific IFN-γ receptor–deficient mice.
In conclusion, we have established for the first time that IFN-γ directly inhibits Hh signaling in (pre)adipocytes in vitro and in adipose tissue in vivo. Our results suggest that the cross-talk between IFN-γ and the Hh-signaling pathway is essential in the maintenance of optimal adipocyte differentiation. The ability to control the plasticity of adipocyte turnover would be a powerful tool in alleviating many complications associated with obesity. That Hh antagonists will one day be used for such purposes in the clinic is perhaps a stretch. However, understanding the regulatory architecture that controls the progenitor cell decision “To differentiate, or not to differentiate?” will be critical to the development of any such therapies. We believe we have added one of the first pieces with which to construct this complex puzzle.