Possible chondroregulatory role of prolactin on the tibial growth plate of lactating rats

Besides calcium accretion in the cortical envelope, a marked increase in the length of long bone was observed in pregnant and lactating rats, and thus the growth plate change was anticipated. Since several bone changes, such as massive trabecular bone resorption in late lactation, were found to be prolactin (PRL)-dependent, PRL may also be responsible for the maternal bone elongation. Herein, we investigated the growth plate change and possible chondroregulatory roles of PRL in the tibiae of rats at mid-pregnancy until 15 days postweaning. We found that the tibial length of lactating rats was increased and was inversely correlated with the total growth plate height, as well as the heights of proliferating zone (PZ) and hypertrophic zone (HZ), but not the resting zone (RZ). Chondrocytes in all zones expressed PRL receptors as visualized by immunohistochemistry, suggesting that the growth plate cartilage was a target of PRL action. Further investigations in lactating rats treated with an inhibitor of pituitary PRL release, bromocriptine, with or without PRL supplement, revealed the PRL-induced decreases in total growth plate height and HZ height from early to late lactation. However, decreases in RZ and PZ heights were observed only in late and mid-lactation, respectively. Thus, this was the first report on the chondroregulatory action of PRL on the growth plate of long bone in lactating rats. The results provided better understanding of the maternal bone adaptation during lactation.     

Proliferation and mRNA expression of absorptive villous cell markers and mineral transporters in prolactin-exposed IEC-6 intestinal crypt cells

During pregnancy and lactation, prolactin (PRL) enhances intestinal absorption of calcium and other minerals for fetal development and milk production. Although an enhanced absorptive efficiency is believed to mainly result from the upregulation of mineral transporters in the absorptive villous cells, some other possibilities, such as PRL-enhanced crypt cell proliferation and differentiation to increase the absorptive area, have never been ruled out. Here, we investigated cell proliferation and mRNA expression of mineral absorption-related genes in the PRL-exposed IEC-6 crypt cells. As expected, the cell proliferation was not altered by PRL. Inasmuch as the mRNA expressions of villous cell markers, including dipeptidylpeptidase-4, lactase and glucose transporter-5, were not increased, PRL was not likely to enhance crypt cell differentiation into the absorptive villous cells. In contrast to the previous findings in villous cells, PRL was found to downregulate the expression of calbindin-D(9k), claudin-3 and occludin in IEC-6 crypt cells, while having no effect on transient receptor potential vanilloid family channels-5/6, plasma membrane Ca(2+)-ATPase (PMCA)-1b and Na(+)/Ca(2+) exchanger-1 expression. In conclusion, IEC-6 crypt cells did not respond to PRL by increasing proliferation or differentiation into villous cells. The present results thus supported the previous hypothesis that PRL enhanced mineral absorption predominantly by increasing transporter expression and activity in the absorptive villous cells.     

Prolactin decreases expression of Runx2, osteoprotegerin, and RANKL in primary osteoblasts derived from tibiae of adult female rats

Hyperprolactinemia caused by physiological or pathological conditions, such as those occurring during lactation and prolactinoma, respectively, results in progressive osteopenia. The underlying mechanisms, however, are controversial. Prolactin (PRL) may directly attenuate the functions of osteoblasts, since these bone cells express PRL receptors. The present study therefore aimed to investigate the effects of PRL on the expression of genes related to the osteoblast functions by using quantitative real-time PCR technique. Herein, we used primary osteoblasts that were derived from the tibiae of adult rats and displayed characteristics of differentiated osteoblasts, including in vitro mineralization. Osteoblasts exposed for 48 h to 1000 ng/mL PRL, but not to 10 or 100 ng/mL PRL, showed decreases in the mRNA expression of Runx2, osteoprotegerin (OPG), and receptor activator of nuclear factor kappaBeta ligand (RANKL) by 60.49%, 72.74%, and 87.51%, respectively. Nevertheless, PRL did not change the RANKL/OPG ratio, since expression of OPG and RANKL were proportionally decreased. These concentrations of PRL had no effect on the mRNA expression of osteocalcin and osteopontin, nor on mineralization. High pathologic concentrations of PRL (1000 ng/mL) may downregulate expression of genes that are essential for osteoblast differentiation and functions. The present results explained the clinical findings of hyperprolactinemia-induced bone loss.     

Impaired body calcium metabolism with low bone density and compensatory colonic calcium absorption in cecectomized rats

An earlier study reported that cecal calcium absorption contributes less than 10% of total calcium absorbed by the intestine, although the cecum has the highest calcium transport rate compared with other intestinal segments. Thus, the physiological significance of the cecum pertaining to body calcium metabolism remains elusive. Herein, a 4-wk calcium balance study in cecectomized rats revealed an increase in fecal calcium loss with marked decreases in fractional calcium absorption and urinary calcium excretion only in the early days post-operation, suggesting the presence of a compensatory mechanism to minimize intestinal calcium wasting. Further investigation in cecectomized rats showed that active calcium transport was enhanced in the proximal colon but not in the small intestine, whereas passive calcium transport along the whole intestine was unaltered. Since apical exposure to calcium-sensing receptor (CaSR) agonists similarly increased proximal colonic calcium transport, activation of apical CaSR in colonic epithelial cells could have been involved in this hyperabsorption. Calcium transporter genes, i.e., TRPV6 and calbindin-D(9k), were also upregulated in proximal colonic epithelial cells. Surprisingly, elevated serum parathyroid hormone levels and hyperphosphatemia were evident in cecectomized rats despite normal plasma calcium levels, suggesting that colonic compensation alone might be insufficient to maintain normocalcemia. Thus, massive bone loss occurred in both cortical and trabecular sites, including lumbar vertebrae, femora, and tibiae. The presence of compensatory colonic calcium hyperabsorption with pervasive osteopenia in cecectomized rats therefore corroborates that the cecum is extremely crucial for body calcium homeostasis.     

Chronic metabolic acidosis upregulated claudin mRNA expression in the duodenal enterocytes of female rats

Previous investigations showed that chronic metabolic acidosis (CMA) increased the paracellular permeability of ion and neutral hydrophilic molecules in the duodenum of rats and small intestinal-like cell lines. Since proteins of the claudin family have been known to regulate the paracellular transport in several epithelia, an increase in the paracellular permeability during CMA may have resulted from changes in the pattern of claudin expression. The present study aimed to investigate the expression profile of 22 claudins in the duodenum of female Sprague-Dawley rats given 1.5% NH(4)Cl for 21 days to induce CMA. Arterial blood gas analysis revealed plasma pH values of 7.40 in normal rats and 7.31 in acidotic rats. Blood chemistry showed increases in the total plasma calcium, free-ionized calcium and magnesium, indicating a typical adaptive response of animals to CMA. RT-PCR demonstrated mRNA expressions of claudin-1 to -12, -14, -15, -17 to -20, -22 and -23 in duodenum of normal rats. Claudin-16 was not expressed in normal duodenum, but was strongly expressed in the kidney. Claudin-13 expression was seen only in the cecum, colon, liver and kidney of mice. After 21-day CMA, mRNA expressions of claudin-2, -3, -6, -8, -11, -12, -14, -19 and -22 were significantly enhanced, whereas expressions of other claudins were not changed. Confocal laser-scanning microscopy demonstrated that duodenal enterocytes of normal rats expressed claudin-3 protein on the paracellular membrane. The distribution of claudin-3 protein along the paracellular membrane was markedly increased in CMA, especially near the apical surface. Our results, therefore, provided novel evidence that 21-day CMA markedly altered claudin profile in the duodenum of rats by upregulating specific claudin expression.     

Symbiotic properties of a chimeric Nod-independent photosynthetic Bradyrhizobium strain obtained by conjugative transfer of a symbiotic plasmid

The lateral transfer of symbiotic genes converting a predisposed soil bacteria into a legume symbiont has occurred repeatedly and independently during the evolution of rhizobia. We experimented the transfer of a symbiotic plasmid between Bradyrhizobium strains. The originality of the DOA9 donor is that it harbours a symbiotic mega-plasmid (pDOA9) containing nod, nif and T3SS genes while the ORS278 recipient has the unique property of inducing nodules on some Aeschynomene species in the absence of Nod factors (NFs). We observed that the chimeric strain ORS278-pDOA9* lost its ability to develop a functional symbiosis with Aeschynomene. indica and Aeschynomene evenia. The mutation of rhcN and nodB led to partial restoration of nodule efficiency, indicating that T3SS effectors and NFs block the establishment of the NF-independent symbiosis. Conversely, ORS278-pDOA9* strain acquired the ability to form nodules on Crotalaria juncea and Macroptillium artropurpureum but not on NF-dependent Aeschynomene (A. afraspera and A. americana), suggesting that the ORS278 strain also harbours incompatible factors that block the interaction with these species. These data indicate that the symbiotic properties of a chimeric rhizobia cannot be anticipated due to new combination of symbiotic and non-symbiotic determinants that may interfere during the interaction with the host plant.     

Osteoblasts express claudins and tight junction-associated proteins

Osteoblasts were previously reported to form tight junctions, which may play an important role in the regulation of ion transport across the epithelial-like bone membrane. However, the evidence for the presence of tight junction-associated proteins in osteoblasts is lacking. We therefore studied the expression of tight junction-associated genes in primary rat osteoblasts and bone tissues. Quantitative real-time PCR showed that osteoblasts expressed ZO-1, -2, -3, cingulin, occludin, claudin-1 to -12, -14 to -20, -22 and -23. By using western blot analyses of selected claudins, expression of claudin-5, -11, -14 and -15, but not claudin-3, were identified in osteoblasts. A confocal immunofluorescent study in undecalcified tibial sections confirmed that claudin-16 was localized on the trabecular surface, normally covered by osteoblasts and bone-lining cells. In addition, immunohistochemical studies in decalcified tibial sections demonstrated the expression of claudin-5, -11, -14, -15 and -16 in bone-lining cells (inactive osteoblasts). Primary osteoblasts cultured in the Snapwell for 19-26 days were found to form a monolayer with measurable transepithelial resistance of approximately 110-180 Omegacm(2), confirming the presence of barrier functions of the tight junction. It was concluded that osteoblasts expressed several tight junction-associated proteins, which possibly regulated ion transport across the bone membrane.