TGF-β regulates numerous classes of genes involved in the cell cycle, apoptosis, cell differentiation, and ECM deposition, and its misregulation leads to physiological disorders that include fibrosis and abnormal wound healing. Regulation of ECM and fibrosis disease-related genes by TGF-β has been proposed to involve binding sites for members of the CTF/NFI family of transcription factors in fibroblastic cell lines (1
). However, whether NFI-C mediates TGF-β signaling in vivo remains unknown. In this work, we performed an mRNA profiling analysis of primary MEFs expressing or not expressing NFI-C that were treated or not with TGF-β1. This led to the identification of NFI-C target genes that prominently featured functions associated with tissue remodeling and development. Given the involvement of tissue remodeling in wound healing, we next analyzed NFI-C functions in this latter process and identified an accelerated early wound healing phenotype in knockout mice that we have linked to abnormal PDGF signaling and faster myofibroblast differentiation.
Upon the treatment of fibroblasts with TGF-β1, the number of genes that were found to be potential targets of NFI-C increased strikingly. This was particularly noticeable after 1 h of treatment, as threefold more genes were misregulated in knockout cells than in untreated cells. This finding is consistent with the observations of Alevizopoulos et al. (1
), who demonstrated that TGF-β1 potentiates transcriptional regulation by NFI-C. Furthermore, when comparing the transcriptional activity of NFI-C in the presence and in the absence of TGF-β1, we noticed an increase in the number of genes that were downregulated by NFIC in TGF-β1-treated cells, and the expression of most of these genes was also inhibited by TGF-β1. This indicates that NFI-C acts mainly as a repressor of genes whose expression is controlled by TGF-β1. Indeed, NFI proteins have been proposed to complex with the Ski oncogene (50
), a repressor of TGF-β signaling that binds to Smad3/4 and represses the expression from promoters that associate with Smad3/4 complexes (29
). As NFI has also been shown to recruit of Ski at promoters (50
), the two mechanisms may act in concert to downregulate the expression of TGF-β target genes.
Analysis of the biological function of misregulated genes suggested that TGF-β1 and NFI-C control genes contribute to the development and function of connective tissues. However, regulation of the expression of ECM genes per se, as previously suggested by low-throughput approaches, does not appear to be the sole function of NFI-C. Analysis of the specific functions of the misregulated genes revealed intermediates of a signaling pathway known to control ECM deposition, tissue remodeling, and/or cell proliferation, such as the aryl hydrocarbon receptor, the PDGF receptor, and the insulin-like growth factor. Therefore, our results implicate NFI-C in the control of signaling pathways that not only mediate ECM deposition but also coordinate more general connective tissue remodeling and repair. This conclusion is supported by the lack of abnormal ECM protein deposition or fibrosis during wound healing in NFI-C knockout mice. Our study revealed an acceleration of the early signaling events that lead to cell recruitment and differentiation within the inflammation phase of wound healing. Indeed, formation of granulation tissue and its later remodeling stage per se were not affected during the healing process, but it is the timing of these events that was altered.
Previous studies have shown that TGF-β1 may act to promote early steps of wound healing, while abnormal signaling was linked to either accelerated or delayed healing (56
). For instance, treatment of an injury with TGF-β1 at the day of wounding was shown to trigger an early infiltration of immune cells and an increase in myofibroblast differentiation, resulting in an accelerated healing and tissue repair (49
). Closure of the wound has been attributed to tissue contraction mediated by myofibroblasts. Differentiation into this contractile form of fibroblasts depends on the de novo expression of the ED-A fibronectin variant (43
). The ED-A variant establishes new interaction with integrins α9β1 and α4β1, and it enhances the TGF-β1-dependent expression of αSMA in fibroblasts (22
). TGF-β is therefore a key regulator of wound closure, as it promotes ED-A FN
alternative splicing, myofibroblast differentiation, and αSMA
The faster early healing process of NFI-C knockout mice is accompanied by the increased recruitment of monocytic cells as well as by a higher expression of ED-A FN
in the early granulation tissue. Furthermore, immunostaining analyses revealed a higher number of myofibroblasts in the wounds of NFI-C-null animals, explaining the enhancement of contraction at the early stage of healing. Later during healing, the kinetics of wound closure decreased in knockout animals, and full closure of the wound coincided with that of the wild-type animals. Thus, the lack of NFI-C displays opposite effects on early or late healing steps, and these effects are similar to those elicited by an early or late topical treatment of the wound with TGF-β1, which accelerates or delays healing, respectively (49
). The finding that, in the absence of the growth factor, NFI-C acts mainly as an antagonist to the changes in gene expression elicited by TGF-β1 readily provides an explanation for this phenomenon. Early in the healing process, just after wounding when TGF-β1 levels are relatively low, the lack of NFI-C will tend to derepress TGF-β1 target genes and thereby accelerate the healing process, as does the ectopic addition of the growth factor. Later in the wound healing process, TGF-β1 expression is increased in mice of both genotypes. Our results indicate that at this stage, NFI-C acts as an agonist of the signaling pathway in the presence of the growth factor. This fits well with the observation that wound closure of wild-type animals catches up with the slowed-down closure of knockout mice later in the healing process, when both the ectopic addition of TGF-β1 and the presence of NFI-C decrease the kinetics of healing. Thus, the dual antagonistic or agonistic role of NFI-C on healing closely parallels the effect of the ectopic addition of TGF-β1, strengthening our conclusion of an implication of the two regulators in a pathway that controls proper timing of this tissue repair process.
While our study cannot formally rule out the possibility of high local concentrations of active TGF-β proteins at the wounds of knockout animals, we rather noted an increase in its expression at later healing stages, which was not altered by the lack of NFI-C. Consistently, the gene expression profiling study provided alternative genes whose altered expression may explain faster healing of the wound of NFI-C-deficient mice. Accelerated wound healing was found to be accompanied by an increase in the expression of both PDGFA and one of its receptors, Pdgfr
α, in the wounded skin of knockout animals. Increased PdgfA
mRNA and protein levels were observed in the skin of knockout mice, even in the normal unwounded skin tissue, indicating that it may be the primary event mediating an early onset of healing. The regulation of PdgfA
expression by the NFI family in skin fibroblasts is consistent with the previous observation of the regulation of PdgfA
gene transcription by the interplay of NFI-X and SP1 in muscle cells. NFI-X was shown to interact with SP1 and to antagonize SP1 binding and activation of the PdgfA
gene promoter (35
). Whether a similar mechanism may also operate to control PdgfA
expression in the skin remains to be ascertained.
The implication of PDGF in the enhancement of an early step of NFI-C−/−
skin wound healing is supported by preliminary data indicating that treatment with an inhibitor of PDGF receptors abolished detectable differences of wound closure by wild-type and knockout animals. Thus, the improved healing observed in null animals may critically depend on the overactivation of the PDGF pathway. PDGF is known to play a central role in the early healing process as a chemotactic signal for monocytes (12
), and the finding of a higher expression of a macrophage marker and an increased macrophage infiltration in the early granulation tissue of knockout animals is consistent with increased signaling by this regulatory cascade. As macrophages are known to secrete PDGFA (38
), increased signaling and an accelerated recruitment of these cells at the site of injury should trigger the induction of a positive feedback loop, resulting from the de novo synthesis of PDGF and from its autocrine action on cells within the granulation tissue.
In addition to its role on the recruitment of monocytes, PDGFA has also been implicated in the acceleration of the recruitment and proliferation of fibroblasts at the site of injury (44
), which in turn may improve repair. Pdgfr
α expression was shown to be under the control of TGF-β1 in a complex fashion. For instance, TGF-β1 downregulated Pdgfr
α in cultures of human fibroblasts, while it increased its expression in sclerodermal fibroblasts (45
). Similarly, our results indicate that the lack of NFI-C increases the expression of PDGFA and of its receptor alpha in wound biopsies in vivo, while Pdgfr
α expression was reduced in cultured fibroblasts. The reasons for such differences have remained unclear, but they may stem from the different behavior of the fibroblasts depending on their origin and/or on the cellular context. In any case, these observations support our conclusion of an antagonistic link between NFI-C and TGF-β signaling in the skin in vivo.
This proposed negative modulation of TGF-β signaling by the basal activity of NFI-C may also explain another phenotype observed in NFI-C−/−
mice: the failure of molar tooth root development and the formation of aberrantly short roots. During this aberrant root development, cells that normally remain on the surface of the dentin matrix become trapped within the matrix, forming a material morphologically similar to osteodentin, a form of dentin deposited during the repair of deep caries (33
). A similar short-root- and osteodentin-forming phenotype is seen in mice overexpressing TGF-β1 from the odontoblast-specific dentin sialophosphoprotein gene promoter (51
). Our model that the loss of NFI-C may potentiate the signaling by low levels of TGF-β1 would indeed predict enhanced TGF-β signaling in the NFI-C−/−
mice that could directly result in the tooth root phenotype seen in these animals.
At present, the mechanisms that regulate the progression of skin wound healing are still poorly understood. PDGF and TGF-β are consistently found at injury sites where they may directly or indirectly control migration, proliferation, and differentiation of macrophages and fibroblasts, but the precise relationships and molecular determinants of these regulatory pathways have remained unclear. Overall, our results shed light on some of these complex regulatory interactions, as they indicate that NFI-C contributes to and modulates the response elicited by both the TGF-β and the PDGFA signaling pathways. Thus, NFI-C may coordinate and interconnect the actions of these two signaling pathways during the wound healing process.
In addition to its role on tissue development and repair, our gene ontology analysis suggested that NFI-C may regulate embryogenesis as well as liver function and hepatic diseases. Consistently, the Ahr
gene that is downregulated in NFI-C-null fibroblasts has been linked to hepatic fibrosis in Ahr−/−
). Healing of the skin is a repair process that resembles the autoregenerative process observed after partial hepatectomy, whereas an exaggerated healing process leads mainly to fibrosis. Other ongoing studies have also implicated NFI-C in the proper timing of another cyclic regenerative process: the hair follicle regrowth during the first hair cycle after birth (G. Plasari, A. Calabrese, and N. Mermod, unpublished data). We did not notice any difference in the hair follicle stage between wild-type and knockout animals at the time of wound excisions, which were performed later after birth. This implies that the wound healing phenotype reported here does not result from differences in hair follicle cycling. Taken together, these findings imply that NFI-C may play a more general function in pathways that regulate tissue regeneration, either after injury or as part of a normal degenerative-regenerative cycle.