Kanogwun Thongchote

Prolactin directly enhances bone turnover by raising osteoblast-expressed receptor activator of nuclear factor κB ligand/osteoprotegerin ratio

Hyperprolactinemia leads to high bone turnover as a result of enhanced bone formation and resorption. Although its osteopenic effect has long been explained as hyperprolactinemia-induced hypogonadism, identified prolactin (PRL) receptors in osteoblasts suggested a possible direct action of PRL on bone. In the present study, we found that hyperprolactinemia induced by anterior pituitary transplantation (AP), with or without ovariectomy (Ovx), had no detectable effect on bone mineral density and content measured by dual-energy X-ray absorptiometry (DXA). However, histomorphometric studies revealed increases in the osteoblast and osteoclast surfaces in the AP rats, but a decrease in the osteoblast surface in the AP+Ovx rats. The resorptive activity was predominant since bone volume and trabecular number were decreased, and the trabecular separation was increased in both groups. Estrogen supplement (E2) fully reversed the effect of estrogen depletion in the Ovx but not in the AP+Ovx rats. In contrast to the typical Ovx rats, bone formation and resorption became uncoupled in the AP+Ovx rats. Therefore, hyperprolactinemia was likely to have some estrogen-independent and/or direct actions on bone turnover. Osteoblast-expressed PRL receptor transcripts and proteins shown in the present study confirmed our hypothesis. Furthermore, we demonstrated that the osteoblast-like cells, MG-63, directly exposed to PRL exhibited lower expression of alkaline phosphatase and osteocalcin mRNA, and a decrease in alkaline phosphatase activity. The ratios of receptor activator of nuclear factor kappaB ligand (RANKL) and osteoprotegerin (OPG) proteins were increased, indicating an increase in the osteoclastic bone resorption. The present data thus demonstrated that hyperprolactinemia could act directly on bone to stimulate bone turnover, with more influence on bone resorption than formation. PRL enhanced bone resorption in part by increasing RANKL and decreasing OPG expressions by osteoblasts.

Transcriptome responses of duodenal epithelial cells to prolactin in pituitary-grafted rats.

Chronic prolactin (PRL) exposure can affect several functions of duodenal epithelia, especially those associated with fluid and electrolyte transport. However, little is known regarding its molecular mechanism. To identify PRL-regulated genes, microarray analysis was performed on RNA samples from duodenal epithelial cells of anterior pituitary (AP)-grafted hyperprolactinemic rats. Herein, we identified 321 transcripts upregulated and 241 transcripts downregulated after 4 weeks of AP transplantation. Results from real-time PCR analyses of 15 selected genes were consistent with the microarray results. Gene ontology analysis demonstrated pleiotropic effects of PRL on several cellular processes, including cellular metabolic process, cell communication and cell adhesion. Interestingly, 17 upregulated transcripts and 12 downregulated transcripts are involved in the transport of ions and nutrients, e.g., Ca(2+), Na(+), K(+), Cl(-) and glucose, thus agreeing with the established action of PRL on electrolyte homeostasis. The present results provided fundamental information for further investigations on mechanism of PRL actions in the intestine.

Impaired bone formation and osteopenia in heterozygous β(IVSII-654) knockin thalassemic mice.

Abstractβ-thalassemia caused by the C→T mutation at nucleotide 654 of the intron 2 (βIVSII-654) results in aberrant splicing of β-globin RNA, leading to an almost absence of β-globin synthesis. Although trabecular and cortical bone loss was previously reported in β-thalassemic mice with deletion of β-globin gene, the microscopic changes in trabecular structure in βIVSII-654 thalassemic mice remained elusive. Here, we investigated the macroscopic and microscopic bone changes in 12-week-old βIVSII-654 knockin thalassemic mice by dual-energy X-ray absorptiometry (DXA) and histomorphometric analysis, respectively. DXA revealed a decrease in bone mineral density in the lumbar vertebrae and tibial metaphysis, but not in the femoral diaphysis, suggesting that βIVSII-654 thalassemia predominantly led to osteopenia at the trabecular site, but not the cortical site. Further histomorphometric analysis of the tibial secondary spongiosa showed that trabecular bone volume was significantly decreased with the expansion of marrow cavity. Decreases in osteoblast surface, osteoid surface, mineral apposition rate, mineralizing surface, and mineralized volume were also observed. Moreover, trabecular bone resorption was markedly enhanced as indicated by increases in the osteoclast surface and eroded surface. It could be concluded that βIVSII-654 thalassemia impaired bone formation and enhanced bone resorption, thereby leading to osteopenia especially at the trabecular sites, such as the tibial metaphysis.

Running exercise alleviates trabecular bone loss and osteopenia in hemizygous β-globin knockout thalassemic mice

A marked decrease in β-globin production led to β-thalassemia, a hereditary anemic disease associated with bone marrow expansion, bone erosion, and osteoporosis. Herein, we aimed to investigate changes in bone mineral density (BMD) and trabecular microstructure in hemizygous β-globin knockout thalassemic (BKO) mice and to determine whether endurance running (60 min/day, 5 days/wk for 12 wk in running wheels) could effectively alleviate bone loss in BKO mice. Both male and female BKO mice (1-2 mo old) showed growth retardation as indicated by smaller body weight and femoral length than their wild-type littermates. A decrease in BMD was more severe in female than in male BKO mice. Bone histomorphometry revealed that BKO mice had decreases in trabecular bone volume, trabecular number, and trabecular thickness, presumably due to suppression of osteoblast-mediated bone formation and activation of osteoclast-mediated bone resorption, the latter of which was consistent with elevated serum levels of osteoclastogenic cytokines IL-1α and -1β. As determined by peripheral quantitative computed tomography, running increased cortical density and thickness in the femoral and tibial diaphyses of BKO mice compared with those of sedentary BKO mice. Several histomorphometric parameters suggested an enhancement of bone formation (e.g., increased mineral apposition rate) and suppression of bone resorption (e.g., decreased osteoclast surface), which led to increases in trabecular bone volume and trabecular thickness in running BKO mice. In conclusion, BKO mice exhibited pervasive osteopenia and impaired bone microstructure, whereas running exercise appeared to be an effective intervention in alleviating bone microstructural defect in β-thalassemia.

High physiological prolactin induced by pituitary transplantation decreases BMD and BMC in the femoral metaphysis, but not in the diaphysis of adult female rats.

High physiological prolactin (PRL) stimulated intestinal calcium absorption and renal calcium uptake in mammals. Previous histomorphometric study revealed a significant increase in bone turnover in the trabecular part of the PRL-exposed long (cortical) bone; however, whole-bone densitometric analysis was unable to demonstrate such effect. We therefore studied differential changes in bone mineral density (BMD) and contents (BMC) of the femoral diaphysis and metaphysis in adult female rats exposed to high PRL induced by anterior pituitary (AP) transplantation. The estrogen-dependent effects of PRL on the femur were also investigated. We found that chronic exposure to PRL had no effect on BMD or BMC of the femoral diaphysis, which represented the cortical part of the long bone. It is interesting that 7 weeks after an AP transplantation, BMD and BMC of the femoral metaphysis were significantly decreased by 8% and 14%, respectively. Ovariectomy (Ovx) for 2, 5, and 7 weeks also decreased BMD and BMC in the femoral metaphysis, but not in the diaphysis. However, the AP transplantation plus Ovx (AP+Ovx) produced no additive effects. Nevertheless, 2.5 microg/kg 17beta-estradiol (E2) supplementation abolished the osteopenic effects of both Ovx and AP+Ovx on the femur. As for the L5-6 vertebrae, BMD and BMC were not affected by PRL exposure, but were significantly decreased by Ovx and AP+Ovx, and such decreases were completely prevented by E2 supplementation. It could be concluded that high physiological PRL induced a significant osteopenia in the trabecular part, i.e., the metaphysis, of the femora of adult female rats in an estrogen-dependent manner. Since PRL had no detectable effect on the vertebrae, the effects of PRL on bone appeared to be site-specific.

Bone microstructural defects and osteopenia in hemizygous βIVSII-654 knockin thalassemic mice: sex-dependent changes in bone density and osteoclast function

β-Thalassemia, a hereditary anemic disorder, is often associated with skeletal complications that can be found in both males and females. The present study aimed to investigate the age- and sex-dependent changes in bone mineral density (BMD) and trabecular microstructure in β(IVSII-654) knockin thalassemic mice. Dual-energy X-ray absorptiometry and computer-assisted bone histomorphometry were employed to investigate temporal changes in BMD and histomorphometric parameters in male and female mice of a β(IVSII-654) knockin mouse model of human β-thalassemia, in which impaired splicing of β-globin transcript was caused by hemizygous C→T mutation at nucleotide 654 of intron 2. Young, growing β(IVSII-654) mice (1 mo old) manifested shorter bone length and lower BMD than their wild-type littermates, indicating possible growth retardation and osteopenia, the latter of which persisted until 8 mo of age (adult mice). Interestingly, two-way analysis of variance suggested an interaction between sex and β(IVSII-654) genotype, i.e., more severe osteopenia in adult female mice. Bone histomorphometry further suggested that low trabecular bone volume in male β(IVSII-654) mice, particularly during a growing period (1-2 mo), was primarily due to suppression of bone formation, whereas both a low bone formation rate and a marked increase in osteoclast surface were observed in female β(IVSII-654) mice. In conclusion, osteopenia and trabecular microstructural defects were present in both male and female β(IVSII-654) knockin thalassemic mice, but the severity, disease progression, and cellular mechanism differed between the sexes.

1,25‐Dihydroxyvitamin D3‐induced intestinal calcium transport is impaired in β‐globin knockout thalassemic mice

Besides being a common haematological disorder caused by a reduction in β‐globin production, β‐thalassemia has been reported to impair body calcium homeostasis, leading to massive bone loss and increased fracture risk. Here, we demonstrated that heterozygous β‐globin knockout thalassemic mice had a lower rate of duodenal calcium absorption compared with the wild‐type littermates, whereas the epithelial electrical parameters, including transepithelial resistance, were not affected, suggesting no change in the epithelial integrity and permeability. Daily subcutaneous injection of 1 µg kg−1 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3] for 3 days enhanced the duodenal calcium absorption in wild‐type, but not in thalassemic mice. Although β‐thalassemia increased the mRNA level of divalent metal transporter‐1, an iron transporter in the duodenum, it had no effect on the transcripts of ferroportin‐1 or the principal calcium transporters. In conclusion, β‐thalassemia impaired the 1,25(OH)2D3‐dependent intestinal calcium absorption at the post‐transcriptional level, which, in turn, contributed to the dysregulation of body calcium metabolism and β‐thalassemia‐induced osteopenia. Copyright