The net effect of TGF-β signaling determines the specification of cell differentiation, growth, and matrix synthesis. It is the product of complex regulatory mechanisms that control secretion, activation, receptor engagement, and intracellular signaling (23
). Tissues synthesize ample amounts of TGF-βs, but only a small fraction needs to be activated to trigger the downstream signaling events. Therefore, the bioavailability of TGF-βs must be tightly controlled by a variety of mechanisms at different levels. Current models for the regulation of TGF-β involve mechanisms for its secretion, extracellular activation in matrix, and antagonism of ligand-receptor interactions (3
). However, besides the well-recognized latent TGF-β pool in the ECM, TGF-β is also abundantly localized in the cytoplasm, especially in the Golgi apparatus and ER prior to secretion (26
). At the same time, the trans-Golgi network (TGN) is a localized concentration of abundant furin convertase activity in the cell. The colocalization of an intracellular TGF-β pool and TGN furin activity raise two important questions: (a) Is furin-dependent maturation of pro-TGF-β regulated?; and (b) Would such regulation be important in controlling TGF-β activity during development? Here, our data suggest that the answer to both questions is yes.
ESL-1, a Golgi protein, binds directly to proTGF-β in the Golgi apparatus and thus limits the processing of the maturation of TGF-β by furin convertase. Esl1–/– cartilage showed increased mature TGF-β2 and p-Smad2. This was correlated with increased ECM deposition and decreased proliferation of chondrocytes. As a result, Esl1–/– mice exhibit chondrodysplasia from embryonic stages. These data suggest not only that ESL-1 plays a role in regulating TGF-β bioavalibility, but also that this mechanism is important for skeletal development.
Furin has been recognized as a housekeeping protein that localizes in the TGN and plays an important role in proteolytically activating large numbers of proprotein substrates in the secretory pathway compartment. These include diverse signaling ligands, receptors, and pathogenic agents (28
). Because the furin-dependent processing affects multiple signaling pathways, simple regulation of furin expression and/or activation might not be sufficient to differentially control activation of diverse signaling pathways in response to environmental or physiological cues. Hence, pathway-specific mechanisms for regulating furin-dependent processing might be one way to control the production and secretion of different morphogens and growth factors. Here, our findings suggest that ESL-1 serves such a novel function by preventing the maturation and secretion of TGF-β.
The expression pattern of ESL-1 overlaps that of TGF-βs in the skeleton and other organs. This supports the specific requirement of ESL-1 for normal TGF-β maturation. ESL-1 function is reminiscent of that of Emilin-1, which acts as a fine-tuning modulator of the TGF-β by regulating TGF-β proteolytic maturation, but in the ECM (20
). Although they act in different cellular compartments, the similarity of ESL-1 and Emilin’s actions strongly suggest that the inhibition of the cleavage of proTGF-β is an important mode for regulating TGF-β bioavailability in general, and alteration of ESL-1 or Emilin function may lead to adverse homeostatic and/or developmental defects. We show that ESL-1’s antagonism of TGF-β function is evolutionarily conserved, since overexpression of xEsl1
led to distinct TGF-β/Nodal-deficient phenotypes in the Xenopus
In mice, the loss of ESL-1 leads to increased TGF-β signaling in the growth plate and a chondrodysplasia phenotype. However, the consequences of TGF-β dysregulation in skeletal development and morphogenesis are complex, though their importance in vivo has been highlighted by different genetic disease phenotypes. ADAMTSL2
mutations in geleophysic dysplasia patients have recently been reported to cause elevated TGF-β secretion and activity, leading to disproportionate short stature and brachydactyly in humans (29
). In contrast, fibrillin1 mutations in Marfan syndrome exhibit increased TGF-β activity but result in tall stature. In earlier skeletal developmental stages, Esl1
is highly expressed in the perichondrium but at low levels in the cartilage. Similarly, fibrillin-1 and ADMTSL2 also exhibit strong expression in the perichondrium, where TGF-βs is abundantly synthesized, suggesting that the perichondrium is particularly important for production and regulation of TGF-β activity and regulation of the growth plate (9
). Previous studies have shown that the TGF-β1 inhibition of chondrocyte proliferation and differentiation in the long bones requires an intact perichondrium (32
). PTHrP plays a central role in maintaining proliferating chondrocytes in an undifferentiating state by relaying TGF-β signaling to the cartilage in a perichondrium-dependent manner (16
). This is consistent with our observation that Esl1–/–
growth plates exhibited delayed terminal differentiation, with increased PTHrP and reduced IHH expression. However, the decreased proliferation in Esl1–/–
chondrocytes may rely on a PTHrP/IHH axis–independent mechanism. Esl1
expression is increased in PZ of growth plate in later skeletal development, suggesting that Esl1
and its TGF-β modulator function are regulated in a temporal-spatial fashion. The perichondrium and growth plate cartilage expresses other critical chondrogenesis signaling factors, including FGF18, Wnt, PTHrP, BMPs, etc. Hence, in those aforementioned TGF-β overactivation genetic models, the temporal-spatial differences in TGF-β activation can lead to diverse signaling interactions with other pathways. This most likely helps to determine the precise regulation of growth plate homeostasis and helps to explain why TGF-β overactivation in different genetic models can exhibit diverse outcomes in the skeletal system.
The functional complexity and context dependence of TGF-β signaling necessitate a similarly diverse and complex set of regulators that control TGF-β bioavailability in both a temporal and spatial fashion. Our results indicate that ESL-1 constitutes a mechanism at the cellular level for controlling proteolytic maturation and Golgi retention. Moreover, ESL-1 is broadly expressed in many other organs and tissues in addition to the skeletal system. Therefore, dysregulation of ESL-1 may be involved in other pathological conditions where dysregulation of TGF-β plays a central role. This includes cancer progression, immune dysregulation, osteoblast/osteoclast coupling, and fibrosis/inflammations. Interestingly, ESL-1 cooperates with PSGL-1 and CD44 to regulate neutrophil rolling, which is critical for recruitment of neutrophils to inflamed tissues (35
). However, whether ESL-1 might specify TGF-β bioavailability during immune responses remains to be studied. Additionally, a posttranslationally modified ESL-1 variant with a unique carbohydrate epitope was specifically overexpressed on the surface of almost all epithelial cancers at precursor stages (21
). Whether the dramatically changed subcellular localization of ESL-1 causes a dysregulation of TGF-β that might contribute to the onset and/or progression of cancer is still unknown. In summary, as what we believe to be a novel intracellular inhibitor for TGF-β bioavailability, ESL-1 may serve as a therapeutic target for regulating TGF-β during different disease processes, such as arthritis and cancer, and the Esl1–/–
mouse will be a useful model to address these other questions.