The Hippo signaling pathway is required for salivary gland development and its dysregulation is associated with Sjogren's syndrome

Abstract

Sjogren's syndrome (SS) is a complex autoimmune disease that primarily affects salivary and lacrimal glands and is associated with high morbidity. Although the prevailing dogma is that immune system pathology drives SS, increasing evidence points to structural defects, including defective E-cadherin adhesion, to be involved in its etiology. We have shown that E-cadherin has pivotal roles in the development of the mouse salivary submandibular gland (SMG) by organizing apical-basal polarity in acinar and ductal progenitors and by signaling survival for differentiating duct cells. Recently, E-cadherin junctions have been shown to interact with effectors of the Hippo signaling pathway, a core pathway regulating the organ size, cell proliferation, and differentiation. We now show that Hippo signaling is required for SMG-branching morphogenesis and is involved in the pathophysiology of SS. During SMG development, a Hippo pathway effector, TAZ, becomes increasingly phosphorylated and associated with E-cadherin and α-catenin, consistent with the activation of Hippo signaling. Inhibition of Lats2, an upstream kinase that promotes TAZ phosphorylation, results in dysmorphogenesis of the SMG and impaired duct formation. SMGs from non-obese diabetic mice, a mouse model for SS, phenocopy the Lats2-inhibited SMGs and exhibit a reduction in E-cadherin junctional components, including TAZ. Importantly, labial specimens from human SS patients display mislocalization of TAZ from junctional regions to the nucleus, coincident with accumulation of extracellular matrix components, fibronectin and connective tissue growth factor, known downstream targets of TAZ. Our studies show that Hippo signaling has a crucial role in SMG-branching morphogenesis and provide evidence that defects in this pathway are associated with SS in humans.

Figure 1

Expression and localization of TAZ during submandibular gland (SMG) embryonic development. (a) Phase microscopy images of CD1 mice epithelial buds (e) and surrounding mesenchyme (m) isolated from embryonic days E12.5-E18.5 during SMG branching morphogenesis. Size bar: 20 μm. (b) Immunoblot of TAZ SMG expression levels at E13.5, E15.5 and E18.5. Bar graph, Fold change in TAZ levels after normalization to GAPDH, *p<0.05. (c) Immunoblot of p-TAZ expression levels at E13.5, E15.5 and E18.5. Bar graph, Fold change in p-TAZ levels after normalization to GAPDH, *p<0.05. (d) Ratio of p-TAZ to total TAZ at E13.5, E15.5 and E18.5. (e) Immunofluorescence localization of SMG TAZ (green) at E13.5, E15.5 and E18.5, counterstained with F-actin (red). (f) Immunofluorescence localization of TAZ (green) at E15.5, counterstained with nuclei (blue).

Figure 2

TAZ interacts with E-cadherin junctional complexes throughout development. (a) E-cadherins were immunoprecipitated from E15.5 and E18.5 SMG tissue lysates and their interaction with TAZ was assessed by immunoblot. Bar graph, Quantification of TAZ/E-cadherin levels. (b) TAZ was immunoprecipitated from E15.5 and E18.5 SMG tissue lysates and its interaction with α-catenin was examined by immunoblot. Bar graph, Quantification of α-catenin/TAZ levels. (c) Immunofluorescence localization of E-cadherin at E.15.5 and E18.5. Size bar: 20 μm. (d) Immunofluorescence localization of α-catenin at E.15.5 and E18.5. Size bar: 20 μm. (e) Immunofluorescence co-localization of α-catenin (red) and TAZ (green) at E15.5 and E18.5. Size bar: 20 μm.

Figure 3

Inhibition of Lats2 kinase perturbs SMG branching morphogenesis. (a) Quantitative PCR of Lats2 transcript levels in non-silenced (NS) or Lats2 silenced (S) glands. **p<0.01. (b) Immunofluorescence localization of BLOCK-iT™ Fluorescent Oligo, a marker of transfection efficiency (green) counterstained with F-actin (red). (c) Phase microscopy images of NS and S SMGs transfected at stage E13.5 at 0h and 48h after Lats2 inhibition. (d) Morphometric analyses of the number of buds at 48h/0h (top) and area of buds at 48h/0h (bottom) of NS and S SMGs. **p<0.01. (e) Immunofluorescence localization of Ki67 (green), counterstained with F-actin (red) and nuclei (blue), in NS and S glands. Size bar: 20μm. (f) Immunofluorescence localization of E-cadherin (green), counterstained with F-actin (red), in NS and S glands. Size bar: 20μm. (g) Immunofluorescence localization of TAZ (green), counterstained with F-actin (red), in NS and S glands. Size bar: 20 μm.

Figure 4

NOD mouse SMGs display dysmorphogenesis and altered expression levels of TAZ and E-cadherin junctional components. (a) Phase microscopy images of SMGs isolated from NOD mice on embryonic days E13.5-E18.5 during SMG branching morphogenesis. Size bar: 20 μm. (b) Immunoblots comparing E-cadherin expression levels in CD1 and NOD SMGs at E13.5, E15.5 and E18.5. Bar graph, Fold change in E-cadherin levels after normalization to GAPDH, *p<0.05. (c) Immunoblots assessing α-catenin expression levels in CD1 and NOD SMGs at E13.5, E15.5 and E18.5. Bar graph, Fold change in α-catenin levels after normalization to GAPDH. (d) Immunoblots comparing IQGAP1 expression levels in CD1 and NOD SMGs at E13.5, E15.5 and E18.5. Bar graph, Fold change in IQGAP1 levels after normalization to GAPDH. (e) Immunoblots comparing TAZ expression levels in CD1 and NOD SMGs at E13.5, E15.5 and E18.5. Bar graph, Fold change in TAZ levels after normalization to GAPDH, *p<0.05. (f) Immunoblots comparing p-TAZ expression levels in CD1 and NOD SMGs at E13.5, E15.5 and E18.5. Bar graph, Fold change in p-TAZ levels after normalization to GAPDH, *p<0.05.

Figure 5

NOD SMGs have aberrant duct formation and disorganized E-cadherin and TAZ. (a) Immunofluorescence localization of E-cadherin (green) in CD1 and NOD mouse SMGs at stage E15.5, counterstained with F-actin (red). Size bar: 20 μm. (b) Immunofluorescence localization comparing TAZ (green) in CD1 and NOD mouse SMGs at stage E15.5, counterstained with F-actin (red). Size bar: 20 μm.

Figure 6

Sjogren's syndrome (SS) compatible labial gland biopsies display loss of polarity, dysregulated Hippo signaling and ECM remodeling. (a) Hematoxylin and Eosin (H & E) staining of a biopsy from an SS non-compatible (Non-SS) patient compared to different degrees of lymphocytic infiltrations (measured as the focus score) of SS patients. Size bar: 50 μm. (b) Immunofluorescence localization of E-cadherin in SS and Non-SS glands, counterstained with DAPI. Size bar: 20 μm. (c) Immunofluorescence analyses of β-catenin in SS and Non-SS glands, counterstained with DAPI. Size bar: 20 μm. (d) Immunofluorescence localization of α-catenin in SS and Non-SS glands, counterstained with F-actin. Size bar: 20 μm. (e) Immunofluorescence localization of IQGAP1 in SS and Non-SS glands, counterstained with DAPI. Size bar: 20 μm. (f) Immunofluorescence localization of TAZ in SS and Non-SS glands (left panel), counterstained with DAPI (right panel). Size bar: 20 μm. (g) Immunofluorescence localization of YAP in SS and Non-SS glands (left panel), counterstained with DAPI (right panel). (h) Immunofluorescence localization of CTGF in SS and Non-SS glands. Size bar: 20 μm. (i) Immunofluorescence localization of fibronectin in SS and Non-SS glands. Size bar: 20 μm.