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

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. TGFβ3 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 TGFβ-deficient mice. These results suggest that CEACAM1 has roles in the initiation of palatal fusion via epithelial cell adhesion

Expression of CEACAM1 in embryonic developing craniofacial tissue.

<p>CEACAM1 expression in the craniofacial region in wild-type embryo at E14.0 (A). Dotted rectangles show the medial edges of the epithelium and submandibular duct. These areas are shown enlarged (B, C). CEACAM1 was expressed in the epithelia of pre-fusion palatal shelf (B, arrowhead), submandibular duct (C) and submandibular gland (D). Scale bar, 200 µm (A), 50 µm (B), 100 µm (C, D).</p

Mouse palatal fusion during palatal development in organ culture and microdissection for microarray analysis.

<p>Opposing palatal shelves had no contact in the midline after 5 h in suspension organ culture (A, B). The medial epithelial seam (MES) was detected in fusing palatal shelves after culture for 24 h (C, D). The MES was disappeared completely after 60 h cultured (E, F). Tissues were excised from the boxed regions at each stage for microarray analysis and qPCR (B, D, F). Scale bar, 500 µm (A, C, E), 50 µm (B, D, F). The qPCR analysis confirmed the expression of <i>Ceacam1</i> mRNA in the boxed regions before, during, and after fusion (G). <i>Ceacam1</i> mRNA expression levels during fusion (17.2±2.1) and after fusion (22.2±2.0) were normalized to before fusion. Bars indicate s.e.m. **P<0.01 compared with control (before fusion).</p

Expression of CEACAM1 and effect of anti-CEACAM1 antibody (mAb CC1) on palate fusion.

<p>CEACAM1 expression in palatal shelves before fusion (A). Enlarged image of dotted rectangle area shown in A (B). CEACAM1 was expressed in the MEE before fusion (B), and was retained in the MES during fusion (C). The MES was not observed after fusion. CEACAM1 was not seen in the center of palatal shelves, but was expressed in the nasal and oral epithelium (D). Arrowheads indicate the distribution of CEACAM1 expression in palatal epithelium (B, C and D). Scale bar, 100 µm (A), 50 µm (B, C, D). To examine the role of CEACAM1, palatal shelves were cultured for 60 h with control (non-specific IgG) and mAb CC1 (anti-CEACAM1 antibody). The frequency of palatal fusion was calculated as the percentage of fused palatal shelves relative to the total number analyzed. The mAb CC1 inhibited palatal fusion (E); the frequency of palatal fusion in the presence of mAb CC1 was 48.3%±6.7%, while control fusion frequency was 79.8%±5.6%. Bars indicate s.e.m. **P<0.01 compared with control.</p

Expression of CEACAM1 in <i>K14-cre</i>;<i>Tgfbr2^fl/fl</i> and <i>Tgfb3^−/−</i> palatal shelves.

<p>CEACAM1 was expressed in the MEE of anterior and posterior regions of palatal shelves in wild-type embryos at E14.5 (A, B). CEACAM1 was clearly expressed in the MEE of the anterior region of the unfused palatal shelf in <i>K14-cre</i>;<i>Tgfbr</i>2<i>^fl/fl</i> (C) and was retained in the oral and nasal triangle of MEE of at E15.5 (D). CEACAM1 was expressed in the epithelium and mesenchyme of both anterior and posterior regions of palatal shelves in <i>Tgfb3</i>^−/− mice at 14.5 (E and F). Scale bar, 50 µm (A–F).</p