We describe domain-specific mutations in the FBN1
gene as the genetic cause for a congenital form of scleroderma, SSS. Whereas prior work has demonstrated genetic alterations and environmental exposures that can contribute to fibrotic phenotypes (49
), we report a genetic alteration that is sufficient to initiate and maintain cutaneous profibrotic programs in people, culminating in a scleroderma phenotype with early onset and complete penetrance. We propose that FBN1
mutations and the consequent perturbation of both microfibrillar assembly and microfibril-integrin interactions contribute to the pathogenesis of SSS, at least in part, through dysregulation of TGFβ signaling.
Several lines of evidence indicate that the defined FBN1
mutations cause SSS. First, the mutations segregated with the phenotype in families and were absent in unaffected family members. The mutation associated with the hybrid phenotype occurred de novo
in the context of sporadic disease. Second, none of the mutations were observed in over 400 ethnically-matched control chromosomes. Third, all of the mutations causing typical SSS involve the creation or removal of a cysteine residue within a domain-type known to be highly dependent upon a defined spacing of eight cysteines for proper folding via intra-domain disulfide linkages (50
). Indeed, the presence of eight cysteines is a defining feature of all TB domains (in fibrillins and LTBPs) throughout evolution (53
). Finally, the mutated tryptophan residue in families 1 and 2 is a key structural residue in TB domains and helps to stabilize the hydrophobic core (52
mutations at the extreme N-terminus of TB4 cause classic MFS, as do substitutions identical to those seen in SSS at orthologous residues in other TB domains (like TB6; ) (55
). TB4 substitutions associated with SSS may cause different effects on domain folding, stability and protein secretion than those seen with MFS-causing substitutions. Furthermore, the presence of an RGD motif in TB4 and the high affinity calcium binding site (Kd
~16nM) formed between TB4 and cbEGF23 (48
) likely confer unique functional properties to this TB domain within fibrillin-1 and hence specific phenotypic consequences upon disruption of domain structure and function.
We also provide evidence that these SSS-specific FBN1
mutations alter cytokine regulation. The contribution of microfibrils to TGFβ regulation is complicated. Studies of MFS have demonstrated that microfibrillar sequestration of the TGFβ LLC limits cytokine activation and signaling (40
). Fibrillin-2 deficient mice show fusion of digits (syndactyly) and were found to have a decreased amount of selected TGFβ superfamily members (e.g. bone morphogenetic protein 7) in developing autopods. This suggests that microfibrils can also serve to concentrate TGFβ ligands in order to achieve the signaling threshold needed to mediate critical morphogenic events (56
). While apparently at odds with events in MFS and perhaps context-specific, the level of TGFβ signaling might simply integrate the divergent roles of microfibrils with other determinants of cytokine bioavailability and activation. Several possibilities may explain why increased TGFβ signaling causes skin fibrosis in SSS but not in MFS. In MFS it is thought that gradual loss of microfibrils (largely during postnatal life) achieves a critical threshold that allows loss of negative regulation of TGFβ activation (40
). Increased activation supports excessive signaling, which leads to changes in gene expression that result in many of the phenotypes in MFS such as pulmonary emphysema, myxomatous changes of the atrioventricular valves, dural ectasia, aortic aneurysm, and muscle hypoplasia and weakness (40
). Consistent with this model, postnatal TGFβ antagonism with systemic delivery of a neutralizing antibody attenuates or prevents many clinical phenotypes in fibrillin-1 deficient mice. In our model of this microfibril-deficient state, decreased TGFβ concentration is offset by increased activation () (40
). An increase in signaling is dependent on ongoing production of TGFβ.
In contrast, given the accumulation of aberrant microfibrillar assemblies in SSS, increased concentration of latent TGFβ may sustain a chronic increased level of TGFβ signaling whether or not the abnormal character of microfibrils promotes increased TGFβ activation. This model is also compatible with the restricted location and small repertoire of mutations in SSS, which is more typical of gain (rather than loss) of function. This same line of reasoning could explain the hybrid phenotype (SSS-MFS) of Tsk mice where the large internal fibrillin-1 duplication (encompassing exon 37) both impairs the context and function of the integrin-interacting RGD sequence and the overall structure and stability of fibrillin-1.
What is the nature of the proposed gain of function in SSS? The unique feature of TB4 is that it encodes the only RGD sequence in fibrillin-1, a motif that mediates cell-matrix interactions via integrin binding. Indeed, the RGD in TB4 of fibrillin-1 is known to interact with α5β1, αvβ3 and αvβ6 integrins (57
). Our in vitro data suggest that SSS mutations impair integrin-mediated cellular events such as the promotion of cellular adhesion and spreading. This does not seem sufficient to initiate fibrosis as such events would also be lost in a microfibrillar deficiency state (i.e. MFS). It is notable that both SSS and SSc associate with excessive accumulation of dermal microfibrillar deposits that concentrate TGFβ in the skin (59
). Increased matrix deposition of abnormal microfibrils may be a consequence of perturbed integrin interactions in SSS. There is precedence for this hypothesis. Takahashi and colleagues showed that upon replacement of the RGD sequence in fibronectin with RGE, cells deposit abnormally short and thickened fibronectin fibrils, reminiscent of the abnormal microfibrillar deposits in SSS (61
). The presumed gain of microfibrillar function in SSS (i.e. concentration of TGFβ LLC) would be imposed early in development and could explain the congenital onset of fibrosis.
It remains unknown whether physiologic or pathologic levels of TGFβ activation accompany the increased concentration of latent TGFβ in the dermis of patients with SSS. The fact that all three integrins that interact with fibrillin-1 (αvβ3, αvβ6 and α5β1) are also known to activate TGFβ (45
) suggests a potential mechanism for enhanced TGFβ activation. Integrins activate TGFβ by two processes, which may act independently or in concert, depending on the tissue context as well as the cell-type involved (37
). First, integrins can simultaneously interact with the latent TGFβ complex (via an RGD sequence in LAP) and other proteins such as matrix metalloproteases and TGFβ receptors to promote TGFβ activation (63
). In a second more mechanical event, integrin binding to the RGD domain of LAPβ1 within the LLC transmits traction forces that conformationally change the LLC and liberate active TGFβ (68
). A mouse with a knock-in mutation of the RGD sequence in LAPβ1 (preventing integrin-mediated activation of TGFβ1) bears striking phenotypic similarity to the TGFβ1 knockout mouse, demonstrating the importance of integrin binding to the LLC in overall TGFβ activation (69
Several studies have implicated aberrant integrin expression or function in SSc and other fibrotic phenotypes. In culture, integrin αvβ3 is upregulated in SSc dermal fibroblasts. Furthermore, its inhibition prevents collagen expression and reverses the myofibroblastic phenotype of SSc fibroblasts in a TGFβ1 – dependent manner (70
). In this light, it is possible (but unproven) that impairment of integrin interaction with fibrillin-1 in SSS induces increased integrin expression and/or bioavailabllity to participate in TGFβ activation, and that this contributes to downstream events including tissue fibrosis.
A number of our observations suggest involvement of basal keratinocytes in the pathogenesis of SSS. These basal keratinocytes showed increased expression of α-SMA and lacked the columnar organization seen in control skin, suggesting the loss of attachments and polarity characteristic of cells undergoing EMT. There was also increased representation of αSMA-positive cells, distinct from the microvasculature, within the peripheral dermis of patients with SSS (). SSS samples also showed αSMA-positive cells occluding small vessels and migrating around their periphery (). These data suggest a contribution of pathologic EMT in SSS, an event plausibly initiated and/or sustained by excessive TGFβ signaling. A specific perturbation of the assembly of microfibrils elaborated by keratinocytes in SSS would explain the normal microfibrillar assemblies found in the deeper dermis of these patients and their lack of manifestations of MFS or fibrosis in other tissues. Human keratinocytes physiologically express integrin α5β1, while they express αvβ6 during wound healing (71
). Although our in vitro experiments using recombinant SSS fibrillin-1 peptides did not reveal altered interaction with VB6 keratinocytes, this does not exclude such perturbations in the context of full-length mutant fibrillin-1 and normal keratinocytes during tissue development or homeostasis in vivo. Informatively, basal keratinocytes have been shown to contribute to pathologic EMT in hypertrophic scar development (72
Our finding of altered microfibrillar assembly in SSc is in keeping with a prior report (73
), but requires further validation in scleroderma-spectrum disorders. The mechanistic basis for this observation remains to be elucidated. Multiple prior reports have described the presence of auto-antibodies to fibrillin-1 in patients with typical presentations of SSc (34
). Although the presence of antibodies that recognize native fibrillin-1 remains controversial (36
), it will be interesting to determine if autoantibodies or another circulating factor in SSc alters the interaction between integrins and fibrillin-1, mimicking the effect of fibrillin-1 mutations in SSS. Modeling of SSS in mice will provide an ideal platform to further interrogate pathogenesis and to test potential therapeutic strategies including TGFβ or EMT antagonists.