Grhl2 regulation of SPINT1 expression controls salivary gland development.

Development of the salivary gland is characterized by extensive branching morphogenesis and lumen formation, the latter of which is closely associated with differentiation into acinar and ductal cells. Although various molecules, including signaling and cell adhesion molecules, have been implicated in salivary gland development, transcription factors (TFs) regulating the expression of those molecules and morphological development of the gland are largely unknown. Here we show that knockdown of the epithelial TF, Grainyhead-like 2 (Grhl2), with siRNA in developing mouse submandibular salivary gland (SMG) cultured ex vivo resulted in retardation of epithelial development. This retardation was concomitant with suppression of gene expression for the cell adhesion molecules, such as E-cadherin and the extracellular protease inhibitor SPINT1, and with the disorganized deposition of the basal lamina protein laminin. ChIP-PCR demonstrated the binding of Grhl2 protein to the Spint1 gene in the SMG. Notably, addition of recombinant SPINT1 protein in cultured SMG overcame the suppressive effects of Grhl2 siRNA on epithelial development and laminin deposition. These findings show that Grhl2 regulation of SPINT1 expression controls salivary gland development.

Effect of hydroxyapatite surface morphology on cell adhesion.

We obtained hydroxyapatite (HAp) materials as a block by mixing HAp nanoparticles and polymer, and then calcining the mixtures. The surface morphology of the HAp materials was tuned by varying heat treatment conditions. After calcining the mixtures at 1200 or 800°C for 4h, the surface morphology of the HAp materials was flat or convexo-concave, respectively. The flat surface morphology, which showed micrometer-ordered grain boundaries, was formed by the aggregation of HAp nanoparticles. On the other hand, the convexo-concave surface morphology resulted from the agglomeration of HAp nanoparticles after heat treatment at 800°C for 4h with nanometer-ordered particle size. We tested cell adhesion to HAp materials with flat or convexo-concave surface morphology and found that cells adhered well to the flat HAp materials but not to the convexo-concave HAp materials. This technique for selectively preparing HAp materials with flat or convexo-concave surface morphology was very easy because we merely mixed commercial HAp nanoparticles with polymer and then calcined the mixtures. As a result, the heat treatment temperature affected the surface morphology of our HAp materials, and their surface morphologies contributed to cell adhesion independently of other material properties.

RNA interference-mediated knockdown of Smad1 inhibits receptor activator of nuclear factor κB ligand expression induced by BMP-2 in primary osteoblasts.

OBJECTIVE: BMP-2 induces osteoblast differentiation and activates osteoclast formation. Here, we investigated the role of Smad1, a molecule that signals downstream of BMP-2, in mediating the effects of BMP-2 on osteoclast differentiation induced by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in osteoblasts. DESIGN: The effects of 1,25(OH)2D3 and BMP-2 in osteoclasts were examined using polymerase chain reaction and Western blotting to measure changes in target gene and protein expression. Immunostaining was carried out to investigate the localization of the vitamin D receptor (VDR) in the nucleus in response to BMP-2. RESULTS: Stimulation with both 1,25(OH)2D3 and BMP-2 resulted in significantly greater osteoclast formation and receptor activator of nuclear factor κB ligand (RANKL) mRNA expression compared to stimulation with 1,25(OH)2D3 alone. In addition, expression of the VDR protein was increased, enhancing the activity of 1,25(OH)2D3. Interestingly, knockdown of Smad1 resulted in reduced osteoclast formation, RANKL mRNA expression, and VDR protein expression compared with control cells. Costimulation with 1,25(OH)2D3 and BMP-2 enhanced VDR localization in the nucleus. CONCLUSIONS: We found that BMP-2 induced Smad1 activation, thereby influencing the localization of VDR in the nucleus in the presence of 1,25(OH)2D3 and resulting in increased RANKL mRNA expression. These effects ultimately resulted in enhanced osteoclast differentiation.

Expression patterns of CD66a and CD117 in the mouse submandibular gland.

The epithelial tissue of the salivary gland consists of the acinar and ductal parts, the latter of which is further divided into the intercalated, striated and excretory duct segments and is the residential site for salivary stem/progenitor cells. In the present study, the expression patterns of two cell surface molecules, CD66a and CD117, were investigated in the adult mouse submandibular glands (SMG) by immunofluorescence microscopy. Combinations of the two molecules differentially marked several types of SMG epithelial cells, including acinar cells (CD66a-intense, CD117-negative), intercalated duct cells (CD66a-intense, CD117-positive), a subset of the striated and excretory duct cells (CD66a-weak, CD117-positive). Most of the CD117-positive ductal cells were negative for cytokeratin 5 and overlapped with the NKCC1-expressing cells. The CD117- and keratin 5-positive cells resided only in the excretory duct were suggested to correspond to the recently identified salivary stem cells. CD66a and CD117 may be useful markers to isolate several cell types consisting of SMG epithelium and to analyze their molecular and cellular nature. Our data also suggest that CD117-expressing epithelial cells of the gland include at least two distinct populations of the stem/progenitor cells.

Regulation of the epithelial adhesion molecule CEACAM1 is important for palate formation.

Cleft palate results from a mixture of genetic and environmental factors and occurs when the bilateral palatal shelves fail to fuse. The objective of this study was to search for new genes involved in mouse palate formation. Gene expression of murine embryonic palatal tissue was analyzed at various developmental stages before, during, and after palate fusion using GeneChip® microarrays. Ceacam1 was one of the highly up-regulated genes during palate formation, and this was confirmed by quantitative real-time PCR. Immunohistochemical staining showed that CEACAM1 was present in prefusion palatal epithelium and was degraded during fusion. To investigate the developmental role of CEACAM1, function-blocking antibody was added to embryonic mouse palate in organ culture. Palatal fusion was inhibited by this function-blocking antibody. To investigate the subsequent developmental role of CEACAM1, we characterized Ceacam1-deficient (Ceacam1(-/-)) mice. Epithelial cells persisted abnormally at the midline of the embryonic palate even on day E16.0, and palatal fusion was delayed in Ceacam1(-/-) mice. TGFß3 expression, apoptosis, and cell proliferation in palatal epithelium were not affected in the palate of Ceacam1(-/-)mice. However, CEACAM1 expression was retained in the remaining MEE of TGFß-deficient mice. These results suggest that CEACAM1 has roles in the initiation of palatal fusion via epithelial cell adhesion.

Membrane-anchored growth factor, HB-EGF, on the cell surface targeted to the inner nuclear membrane.

Heparin-binding EGF-like growth factor (HB-EGF) is synthesized as a type I transmembrane protein (proHB-EGF) and expressed on the cell surface. The ectodomain shedding of proHB-EGF at the extracellular region on the plasma membrane yields a soluble EGF receptor ligand and a transmembrane-cytoplasmic fragment (HB-EGF-CTF). The cytoplasmic domain of proHB-EGF (HB-EGF-cyto) interacts with transcriptional repressors to reverse their repressive activities. However, how HB-EGF-cyto accesses transcriptional repressors is yet unknown. The present study demonstrates that, after exposure to shedding stimuli, both HB-EGF-CTF and unshed proHB-EGF translocate to the nuclear envelope. Immunoelectron microscopy and digitonin-permeabilized cells showed that HB-EGF-cyto signals are at the inner nuclear membrane. A short sequence element within the HB-EGF-cyto allows a transmembrane protein to localize to the nuclear envelope. The dominant-active form of Rab5 and Rab11 suppressed nuclear envelope targeting. Collectively, these data demonstrate that membrane-anchored HB-EGF is targeted to the inner nuclear membrane via a retrograde membrane trafficking pathway.

Hedgehog peptide promotes cell polarization and lumen formation in developing mouse submandibular gland.

Tube formation of the developing mouse submandibular salivary gland (SMG) begins at embryonic day (E) 14. The SMG of Sonic hedgehog (Shh) null mice was recently shown to fail to progress to stages beyond around E14. Here, we examined the effects of Shh peptide on tube formation of SMG explants. When the SMG rudiments from E14 mice were cultured, terminal buds of glands treated with Shh peptide formed the acini-like structure with a lumen whereas those of control glands remained as cell masses. In the acini-like terminal buds of the treated glands, tight junction proteins of ZO-1 and claudin-3 delineated the lumen and the apical membrane protein aquaporin-5 accumulated at the luminal cell surfaces. Moreover, laminin-5 deposition at the basal lamina region of terminal buds was accelerated in treated glands. It is suggested that hedgehog signaling promotes lumen formation and cell polarization of developing SMG epithelium.

Expression patterns of claudin family of tight junction membrane proteins in developing mouse submandibular gland.

Here, we investigated the expression of the claudin family of tight junction transmembrane proteins in the developing mouse submandibular gland. Data obtained by reverse transcriptase-polymerase chain reaction, Western blot, and immunofluorescence microscopy showed the expression and localization of claudin-3 to -8, -10, and -11 at epithelial tight junctions. Examination of the glands taken from embryonic day (E) 14, E16, and newborn mice revealed differential expression patterns of these claudins in the developing epithelium. Claudin-3, -5, and -7 were expressed in all of the luminal epithelial cells of the ducts at all of the developmental stages examined and in those of terminal tubules at E16 and later. Claudin-4 was expressed mainly in the ducts at all the developmental stages. The expression of claudin-6 and -8 was also restricted to the ducts at E14 and E16; but after birth, the former was undetectable, whereas the latter was expressed in both the ducts and terminal tubules. Claudin-10 and -11 were detectable mainly in the terminal tubules at E16 and later. In addition to being found in the epithelium, claudin-5 was also expressed in certain mesenchymal cells, probably endothelial cells. These results will provide a valuable resource for further investigation of tubulogenesis and physiological regulation of claudin-based tight junctions.

Tissue interaction mediated by neuregulin-1 and ErbB receptors regulates epithelial morphogenesis of mouse embryonic submandibular gland.

Dimerization and activation of ErbB receptors by their ligands play crucial roles in organogenesis. Epithelial morphogenesis of embryonic mouse submandibular gland (SMG) has been shown to depend on intraepithelial signaling mediated by the epidermal growth factor (EGF) family of molecules and the EGF receptor (ErbB1). Here, we report on the neuregulin (NRG) -1 protein and its receptors ErbB2 and ErbB3 in the developing SMG. The expression of these molecules was demonstrated by reverse transcriptase-polymerase chain reaction and Western blot analysis. Immunofluorescence microscopy showed that the two ErbB receptors as well as ErbB1 were expressed mainly in the epithelium, whereas NRG-1 was exclusively found in the mesenchyme. Epithelial morphogenesis was retarded by anti-NRG-1 neutralizing antibody and promoted by recombinant NRG-1 protein. We suggest that, in the developing SMG, both mesenchyme-derived NRG molecules and epithelium-derived EGF molecules regulate ErbB signaling in the epithelium to participate in tissue morphogenesis.

Role of Sonic hedgehog signaling in epithelial and mesenchymal development of hair follicles in an organ culture of embryonic mouse skin.

Studies with gene knockout mice have shown that Sonic hedgehog (Shh) is required for early development of hair follicles, but the role of this gene in the late stages of follicle development is not clear. By using an organ culture system of embryonic mouse skin, the role of Shh signaling in the early and late stages of follicle development was investigated. In the early stage of follicle development, the downward growth of the follicular epithelium was suppressed by cyclopamine, an inhibitor of Shh signaling, and accelerated by recombinant Shh. In addition, cyclopamine impaired dermal papilla formation, accompanied by the rearrangement of papilla cells, but not the elongation of the follicular epithelium at the later stage. These results suggest that Shh signaling is required for the proliferation of epithelial cells in the early development of hair follicles and for the morphogenetic movement of mesenchymal cells at the later stage of follicle development.

Establishment of cadherin-based intercellular junctions in the dermal papilla of the developing hair follicle.

During hair follicle development, mesenchymal cells aggregate to form the dermal papilla with hair-inducing activity. However, the cellular mechanisms underlying the aggregative behavior of dermal papilla cells are less known. The present study demonstrates that cadherin-based intercellular junctions interconnect dermal papilla cells in developing hair follicles of mice. It is shown that as mesenchymal cells aggregate to be surrounded by epithelium in developing hair follicles, cadherin-11 comes to exhibit the dotted patterns of distribution. The appearance of the dot-like distribution of the molecule is concomitant with the formation of intercellular junctions in the mesenchymal aggregate, which make a tightly packed population of cells with little extracellular space. At later stages of the development, although extracellular space reappears in the dermal papilla, the cells remain interconnected by well-developed intercellular junctions, where cadherin-11 as well as beta-catenin is localized. Taking into consideration the normal hair development in cadherin-11 mutant mice, it might be that multiple cadherins are responsible for the establishment of intercellular junctions in the dermal papilla and serve to maintain the aggregative behavior of the cells.