Expression and signal transduction of calcium-sensing receptors in cartilage and bone.

We previously showed that Ca2+-sensing receptors (CaRs) are expressed in chondrogenic RCJ3.1C5.18 (C5.18) cells and that changes in extracellular [Ca2+]([Ca2+]o) modulate nodule formation and chondrogenic gene expression. In the present study, we detected expression of CaRs in mouse, rat, and bovine cartilage and bone by in situ hybridization, immunocytochemistry, immunoblotting, and RT-PCR; and we tested the effects of CaR agonists on signal transduction in chondrogenic and osteogenic cell lines. In situ hybridization detected CaR transcripts in most articular chondrocytes and in the hypertrophic chondrocytes of the epiphyseal growth plate. Expression of CaR transcripts was weak or absent, however, in proliferating and maturing chondrocytes in the growth plate. In bone, CaR transcripts were present in osteoblasts, osteocytes, and bone marrow cells, but rarely in osteoclasts. A complementary DNA was amplified from mouse growth plate cartilage, which was highly homologous to the human parathyroid CaR sequence. Immunocytochemistry of cartilage and bone with CaR antisera confirmed these findings. Western blotting revealed specific bands (approximately 140-190 kDa) in membrane fractions isolated from growth plate cartilage, primary cultures of rat chondrocytes, and several osteogenic cell lines (SaOS-2, UMR-106, ROS 17/2.8, and MC3T3-E1). InsP responses to high [Ca2+]o were evident in C5.18 cells and all osteogenic cell lines tested except for SaOS-2 cells. In the latter, high [Ca2+]o reduced PTH-induced cAMP formation. Raising [Ca2+]o also increased intracellular free [Ca2+] in SaOS-2 and C5.18 cells. These studies confirm expression of CaRs in cartilage and bone and support the concept that changes in [Ca2+]o may couple to signaling pathways important in skeletal metabolism.

Glucocorticoid deficiency delays stratum corneum maturation in the fetal mouse.

The stratum corneum (SC) matures during late gestation in man and other mammals. Using the fetal rat as an experimental model, we have previously shown that glucocorticoids given in pharmacologic doses accelerate fetal SC maturation and barrier formation. To determine whether glucocorticoids are required for normal SC maturation, we examined the epidermal morphology of glucocorticoid-deficient (C-) murine pups, derived from matings of mice homozygous for null mutations of the corticotropin-releasing hormone alleles. In control pups on day 17.5 of gestation (term is 19.5 d), a multilayered SC was present and neutral lipid deposition in a membrane pattern was observed using Nile red fluorescence histochemistry. Ultrastructurally, mature lamellar unit structures predominate in the SC intercellular domains. In contrast, in C-pups only a single layer of SC was evident on day 17.5, and secreted lamellar material was not organized into mature lamellar structures. Furthermore, the expression of structural proteins necessary for cornified envelope formation, involucrin, loricrin, and filaggrin, and the activity of the lipid synthetic enzymes beta-glucocerebrosidase and steroid sulfatase, markers of barrier maturation, were reduced in day 17.5 C-pups. C-pups derived from pregnancies supplemented with physiologic amounts of cortisone, however, display normal SC ultrastructure on day 17.5 of gestation. Furthermore, at birth, both control and C-pups exhibit a multilayered SC replete with mature lamellar membrane structures. These data demonstrate that fetal glucocorticoid deficiency delays SC maturation, and suggests that normal levels of glucocorticoids are not absolutely required for SC development.

Lenten cell: ultrastructure, absorptive properties, and enzyme expression of a novel type of cell in the newborn and suckling pig intestinal epithelium.

BACKGROUND: The small intestinal epithelium is made up of columnar absorptive enterocytes and a smaller number of specialized non-absorptive cells, including goblet cells, enteroendocrine cells, M cells, cup cells, and tuft cells. During a study on milk protein absorption in newborn pigs, we identified an enterocyte that showed no uptake of milk proteins and that could be found only in the jejunum and ileum of pigs during the first 2 weeks of life. We call this previously undescribed enterocyte the lenten cell. METHODS: We used light microscopy and transmission electron microscopy in conjunction with immunolabelling and cytochemical techniques to determine the occurrence, ultrastructure, absorptive properties, and brush border hydrolase expression of lenten cells. RESULTS: Lenten cells constituted approximately 1-2% of the villous epithelium. They were seen in newborn and suckling pigs 1-9 days of age, but were not found in weaned pigs. Morphologically, lenten cells were spindle- or wineglass-shaped, with a ventrally sited nucleus and an electron-dense cytoplasm with numerous cytokeratin filaments. Lenten cells had a normal brush border with microvilli that were slightly thicker than those of absorptive enterocytes, but they did not express the brush border hydrolases lactase, aminopeptidase N, and alkaline phosphatase. Lenten cells did not endocytose milk proteins or horseradish peroxidase, but contained some endocytic or secretory vacuoles and a few dense granules. CONCLUSIONS: No role for lenten cells has been identified in this study, but presence of these cells during the neonatal period, when growth and differentiation of the gastrointestinal tract is at a peak, clearly suggests that lenten cells may play a role in this process.

Uptake of maternal immunoglobulins in the enterocytes of suckling piglets: improved detection with a streptavidin-biotin bridge gold technique.

In ungulates, intestinal absorption of maternal immunoglobulins from colostrum plays a vital role in the acquisition of passive immunity during early neonatal life. In the present study we used post-embedding colloidal gold labeling to examine the intracellular localization of IgG in the jejunal enterocytes of miniature piglets suckled for 2 hr. Quantitation of the immunolabeling revealed that the most sensitive technique for IgG detection was the streptavidin bridge-gold technique. In this method, the LR White-embedded sections were labeled sequentially with biotinylated anti-porcine IgG, streptavidin, and biotinylated BSA conjugated to 10-nm colloidal gold. With this approach, we found the following sequence of maternal IgG accumulation: passage of IgG from colostrum through the brush border; binding to the apical plasma membrane; uptake in noncoated pits and invaginations; transport in endocytotic vesicles; and accumulation in granules in the apical cytoplasm.