**5. Impaired NF-**κ**B Signalling Activated by EDA-A1**/**EDAR in pSS Salivary Glands**

In addition to its role in mediation inflammation, NF-κB is also essential for developing the epidermal derivatives, hair, nails, and SGs [62]; a series of molecular signals is now well defined, beginning with the binding of ectodysplasin (EDA-A1) to the EDA-receptor (EDAR), components of the tumour necrosis factor α (TNFα)-related signalling pathway [62]. EDA-A1 signalling is recognized as an important evolutionarily conserved pathway regulating the formation and patterning of vertebrate skin appendages, including SGs [63]. When these genes show mutations, a condition known as hypohidrotic (or anhidrotic) ectodermal dysplasia (HED/EDA) occurs [64]. The NF-κB pathway that mainly impinges on EDA-A1/EDAR-dependent SGs branching morphogenesis is the canonical NF-κB activation cascade [65].

Over the last years, great progress has been made in identifying the key molecular regulators controlling NF-κB activation, and a repertoire of crucial self-regulators ensuring the termination of NF-κB responses has been identified [66]. Interestingly, this well-orchestrated biological process may undergo alterations [33,34,67] and, consequently, deregulated NF-κB activation contributes to the autoimmune diseases pathogenesis, characterized by an intense inflammatory response [34,67]. As a matter of fact, NF-κB was demonstrated to play a salient role in the pathological development of pSS, correlated with the intense chronic inflammation findings in this disease [17,18,25,26,48]. In this context, several studies conducted on SGEC derived from pSS patients investigated the mechanism-of-action of the NF-κB cascade and performed target identification in the deregulated inflammatory situation. Recent findings demonstrated that EDA-A1 induces several genes involved in the synthesis of the NF-κB pathway molecules, including the feedback inhibitors IκBα and TNFAIP3. IκBα is known to be expressed in hair placodes and SGs [68], and TNFAIP3, a key negative feedback regulator of the NF-κB signalling cascade, plays a role in the EDA-A1, EDAR and EDAR-associated death domain (EDARADD) genes control, which results mutated in HED/EDA [69]. Therefore, recently, chemokines have been revealed as immediate target genes of the EDA/NF-κB pathway, leading to modulation of the multiple signalling pathways implicated in skin appendage development; when this scheme is deregulated, an inflammatory process may be induced [69]. Against this background, a recent study has investigated the EDA-A1 and EDAR genes and proteins expression in pSS SGs, showing that TNFAIP3 is deregulated in pSS SGEC. This results in an increased and excessive EDA-A1/EDAR gene and protein expression in pSS SGEC that determines a correlated high induction of NF-κB [70] (Figure 3). Furthermore, TNFAIP3 gene knockdown performed on healthy SGEC, through the application of the

siRNA gene silencing technology, determined an over-activation of the EDA-A1/EDAR expression and consequently NF-κB nuclear translocation and activation [70] (Figure 3). The authors have shown that, in pSS SGEC, NF-κB is activated downstream of EDA-A1/EDAR signalling and after transfecting pSS SGEC with the mutated form of the regulatory protein IκBα, the EDA-A1/EDAR-NF-κB signalling pathway was affected in SGs, suggesting that the IκBα-dependent canonical NF-κB cascade was active in pSS SGEC [70]. This recent discovery suggests that the pathways involved in ectodermal development and inflammation may be fundamentally the same, but lead to target gene activation depending on the cell type and/or on the specific pathological condition features. The implication of the NF-κB pathway in development was a very surprising finding, because it is involved primarily in TNF-α receptors-mediated inflammation and immunity; now, the recurrent question is how cells can distinguish between the NF-κB pathway activation signals, as well as how specific target genes activation is precisely and independently controlled during developmental or inflammatory events.

**Figure 3.** Schematic overview of the EDA/EDAR/canonical NF-κB pathway. The EDA isoform of the TNF-α family member Ectodysplasin interacts with its receptor EDAR leading to the recruitment of EDARADD (death domain adaptor); in turn, this complex activates the IKK complex. The IKK complex phosphorylates IκBα, that undergoes ubiquitylation and proteasomal degradation, inducing nuclear translocation of the NF-κB heterodimer RelA/p50 that triggers the transcription of pro-inflammatory genes, including those that encode the negative regulators IκBα and TNF-α-induced protein 3 (TNFAIP3). EDA: Ectodysplasin-A; EDAR: Ectodysplasin-A Receptor; EDARADD: Ectodysplasin-A receptor-associated associated death domain.
