Natural killer cells, bone, and the bone marrow: studies in estrogen-treated mice and in congenitally osteopetrotic (mi/mi) mice.

Mice lose natural killer cells after 6 weeks of treatment with 17 beta-estradiol. We here demonstrate that the same protocol leads to loss of genetic resistance to bone marrow transplantation and to significant osteoproliferation with loss of bone marrow. We also show that mice with reduced marrow because of congenital osteopetrosis are deficient in natural killing. These findings are consistent with previous evidence that natural killing and genetic resistance to bone marrow transplantation are dependent upon the marrow. Temporal studies of bone histology and radiology during and after treatment with estrogen reveal that alterations in natural killing proceed more rapidly than changes in bone marrow volume. These studies also demonstrate that estrogens induce osteoproliferation only at endosteal surfaces that are adjacent to hematopoietic marrow. From these observations, we conclude that estrogens do not reduce natural killer cells simply by reducing the volume of bone marrow. Estrogens may instead have an effect on bone marrow. Estrogens may instead have an effect on bone marrow cells that leads both to osteoproliferation and to a deficiency of marrow-dependent cells.     

Impaired osteoclast formation in bone marrow cultures of Fgf2 null mice in response to parathyroid hormone

Fibroblast growth factor (FGF)-2 and parathyroid hormone (PTH) are potent inducers of osteoclast (OCL) formation, and PTH increases FGF-2 mRNA and protein expression in osteoblasts. To elucidate the role of endogenous FGF-2 in PTH responses, we examined PTH-induced OCL formation in bone marrow cultures from wild type and mice with a disruption of the Fgf2 gene. FGF-2-induced OCL formation was similar in marrow culture from both genotypes. In contrast, PTH-stimulated OCL formation in bone marrow cultures or co-cultures of osteoblast-spleen cells from Fgf2-/mice was significantly impaired. PTH increased RANKL mRNA expression in osteoblasts cultures from both genotypes. After 6 days of treatment, osteoprotegerin protein in cell supernatants was 40-fold higher in vehicle-treated and 30-fold higher in PTH-treated co-cultures of osteoblast and spleen cells from Fgf2-/mice compared with Fgf2+/+ mice. However, a neutralizing antibody to osteoprotegerin did not rescue reduced OCL formation in response to PTH. Injection of PTH caused hypercalcemia in Fgf2+/+ but not Fgf2-/mice. We conclude that PTH stimulates OCL formation and bone resorption in mice in part by endogenous FGF-2 synthesis by osteoblasts. Because RANKL- and interleukin-11-induced OCL formation was also reduced in bone marrow cultures from Fgf2-/mice, we further conclude that endogenous FGF-2 is necessary for maximal OCL formation by multiple bone resorbing factors.     

Bone surface structure in osteopetrotic grey lethal (gl/gl) and microphthalmic (mi/mi) mutant mice as revealed by scanning electron microscopy

The surface structure of bone from two genetically distinct osteopetrotic strains of mice, grey lethal (gl) and microphthalmic (mi) has been examined by scanning electron microscopy. Although both conditions produce a classical osteopetrolic phenotype the means by which this is achieved is quite different. gl mice appear to retain woven bone and show no evidence of resorption: mi mice show evidence of imperfect remodelling. These results are in accordance with what is already known of osteoclast structure and function in these mutations.     

Rapid activation of the medullary bone osteoclast cell surface by parathyroid hormone

Quantitative transmission electron microscope methods were used to determine the response of functionally inactive avian medullary bone osteoclasts to parathyroid hormone (PTH). Egg-lying Japanese quail were used during a period of the egg cycle when medullary bone was not being resorbed for egg shell calcification and when medullary bone osteoclasts were functionally inactive. Ruffled borders adjacent to bone surfaces were rarely, if ever, found on these cells. 20 min after the administration of PTH, over 70% of the osteoclast profiles had ruffled borders adjacent to bone surfaces. These ruffled borders were bounded by filamentous-rich "clear zones" and resembled ruffled borders found on functionally active cells. There was also a marked increase in plasma calcium levels after PTH administration. This study demonstrates that PTH stimulates the de novo generation of ruffled borders on osteoclasts in vivo and suggests that osteoclasts may be involved in the acute regulation of calcium metabolism by exogenous PTH.     

Influences of osteoclast deficiency on craniofacial growth in osteopetrotic (op/op) mice

It is well known that the defect in bone resorption in osteopetrotic (op/op) mice brings about deformation of the cranium and failure of tooth eruption. However, the influences on longitudinal growth of the craniofacial skeleton have not been elucidated. This study was thus conducted to examine craniofacial morphology and longitudinal changes in the op/op mice by means of morphometric analysis with lateral cephalograms. Lateral cephalograms, taken every 10 days from 10- to 90-day-old mice, were analyzed on a personal computer for 11 measurement items. For the nasal bone region, the most prominent differences were found between the op/op and normal mice. The anterior cranial base and occipital bone height presented almost equivalent growth changes in both the op/op and normal mice. The size of mandible, meanwhile, was significantly smaller in the op/op mice than in the normal controls. The gonial angle was also significantly larger in the op/op mice than in the normal mice throughout the experimental period. Thus, substantial differences in craniofacial growth were demonstrated in various areas of the craniofacial complex, which are assumed essentially due to the lack of osteoclastic bone resorption during growing period. Since the difference became more prominent in the anatomic regions relevant to the masticatory functions, it would be a reasonable assumption that reduced masticatory function is also a key determinant for the less-developed craniofacial skeleton in the op/op mouse.     

Congenital osteoclast deficiency in osteopetrotic (op/op) mice is improved by ovariectomy and orchiectomy.

We examined the changes in the appearance of osteoclasts in the femora of ovariectomized (OVX) or orchiectomized (ORX) op/op mice. Osteoclasts on the trabecular bone surface of the OVX or ORX op/op mice significantly increased in number seven or eight times in comparison with sham-operated op/op mice. Furthermore, TRAP-positive cells increased about four times in 100-week-old females and males, compared with sham-operated groups. These results have indicated that a sex hormone reduction due to OVX or ORX induces prominent recruitment of osteoclasts in op/op mice.     

Effects of 1,25 dihydroxyvitamin D on osteoclast number and cytochemistry in normal and osteopetrotic (os) rabbits

Osteoclast-mediated bone resorption is increased in response to 1,25 dihydroxyvitamin D (1,25[OH]2D or calcitriol). Osteopetrosis is a metabolic bone disease characterized by defective, osteoclast-mediated bone resorption, which co-exists with elevated serum 1,25-(OH)2D levels in some osteopetrotic children and animals. We examined the effects of high doses of calcitriol on osteoclast number and cytochemistry in both normal and osteopetrotic (os) rabbits. Calcitriol was continuously infused at doses of 0.5, 2.5, or 25 micrograms/kg/day via subcutaneously implanted osmotic minipumps for a period of 7 days. Following treatment, the proximal tibial metaphyses were processed for histomorphometric and cytochemical analyses. Sections were stained for tartrate-resistant acid phosphatase (TrAP) or acid ATPase (TraATPase). Osteoclasts were significantly reduced in untreated os rabbits compared with age-matched normal littermates between birth and 3 weeks of age (41-46% of normal). Whereas most normal osteoclasts (85%) stained heavily for TrAP or TraATPase, less than half of os osteoclasts were heavily stained for these acid hydrolases. Infusions of 1,25(OH)2D resulted in elevations of osteoclast numbers in both normal and os rabbits, but the number of osteoclasts remained significantly lower in mutants than in normal littermates at any given dose. Calcitriol infusions also resulted in a significant increase in the percentage of os osteoclasts staining heavily for TrAP and TraATPase. These results suggest that in response to 1,25(OH)2D normal osteoclasts increase their production of acid hydrolases before increasing cell numbers and that, in spite of high levels of endogenous calcitriol, os rabbits can respond to exogenous 1,25(OH)2D as evidenced by increased osteoclast number and cytochemical staining, even though these osteoclasts fail to resorb the excess skeletal matrix.(ABSTRACT TRUNCATED AT 250 WORDS)     

Defective osteoclast differentiation and function in the osteopetrotic (os) rabbit

We tested the ability of normal osteoclast progenitors found in neonatal liver and bone marrow to develop into functional osteoclasts when co-cultured with metatarsals from newborn osteopetrotic rabbits; the latter inherit an osteoclast incompetence resistant to cure by bone marrow transplantation. This system, developed by Burger and colleagues, has been shown to produce normal, functional osteoclasts when used with normal metatarsals. Our study tested the competence of the mutant skeletal microenvironment for differentiation of normal osteoclasts. Mutant and normal metatarsals were cultured alone or with normal liver, spleen, or bone marrow for up to 14 days. All normal cultures possessed a marrow cavity and contained numerous osteoclasts with cytochemical characteristics (tartrate-resistant acid phosphatase) of active cells. Mutant metatarsals co-cultured with normal spleen, liver, or bone marrow failed to develop a marrow cavity (evidence in itself of reduced bone resorption) and had osteoclasts reduced in both numbers and cytochemically detectable activity. Similar metatarsal cultures of an osteopetrotic rat mutation (incisors--absent) curable by bone-marrow transplantation exhibited marrow cavity development in mutant metatarsals co-cultured with normal spleen. These data suggest that the skeletal environment of osteopetrotic rabbits contains an inhibitor or lacks a promoter of osteoclast differentiation and function.     

Lack of the bone remodeling in osteopetrotic (op/op) mice associated with microdontia.

Osteopetrosis is an inherited metabolic disease characterized by an excessive accumulation of bone. This is associated with an osteoclast deficiency. Osteopetrosis is always accompanied by the failure and/or delay of tooth eruption. The present study was conducted to examine in detail the morphological and histological changes of growth of the third molars in the osteopetrosis (op/op) mouse. At the age of 10 days, normal and op/op mice showed no detectable difference in the shape of the third molar follicles. However, the third molars in the op/op mouse became obscured by the proliferation of neighboring bone trabeculae. Moreover, no tartrate-resistant acid phosphatase-positive cells were detected on the bone surfaces of 10-day-old op/op mice. Ankylosis between the root dentin and proliferating bone trabeculae was a common feature in the 20- and 30-day-old op/op mice. The third molars erupted into the oral cavity before the age of 30 days in normal mice, and the crowns, roots, and periodontal ligaments appeared well developed. Throughout the experiment, it seemed that the primary cause of the microdontia and ankylosis of the developing root in the mutant mouse was a deficiency of osteoclasts, with attendant lack of bone remodeling.     

IL-6 is not required for parathyroid hormone stimulation of RANKL expression, osteoclast formation, and bone loss in mice

Continuous elevation of parathyroid hormone (PTH) increases osteoclast precursors, the number of osteoclasts on cancellous bone, and bone turnover. The essential molecular mediators of these effects are controversial, however, and both increased receptor activator of NF-kappaB ligand (RANKL) and IL-6 have been implicated. The goal of these studies was to determine whether continuous elevation of endogenous PTH alters IL-6 gene expression in vivo and whether IL-6 is required for PTH-induced bone loss. To accomplish this, we generated transgenic mice harboring a luciferase reporter gene under the control of IL-6 gene regulatory regions to allow accurate quantification of IL-6 gene activity in vivo. In these mice, induction of secondary hyperparathyroidism using a calcium-deficient diet did not alter IL-6-luciferase transgene expression, whereas RANKL mRNA expression was elevated in bone tissue. Moreover, secondary hyperparathyroidism induced an equivalent amount of bone loss in wild-type and IL-6-deficient mice, and PTH elevated RANKL mRNA and osteoclast formation to the same extent in bone marrow cultures derived from wild-type and IL-6-deficient mice. These results demonstrate that IL-6 is not required for the osteoclast formation and bone loss that accompanies continuous elevation of PTH.     

Stimulation by defined parathyroid hormone fragments of cell proliferation in skeletal-derived cell cultures.

We have reported previously that parathyroid hormone (PTH) acts on cultured bone cells to stimulate creatine kinase (CK) activity and [3H]thymidine incorporation into DNA via phosphoinositide turnover, in addition to its other actions via increased cyclic AMP production. We also found that mid-region fragments of PTH stimulate [3H]thymidine incorporation into avian chondrocytes. In the present study of mammalian systems, we demonstrate differential effects of defined synthetic PTH fragments on CK activity and DNA synthesis, as compared with cyclic AMP production, in osteoblast-enriched embryonic rat calvaria cell cultures, in an osteoblast-like clone of rat osteosarcoma cells (ROS 17/2.8) and in chondroblasts from rat epiphysial cartilage cell cultures. Unlike full-length bovine (b)PTH-(1-84) or the fully effective shorter fragment human (h)PTH-(1-34), fragments lacking the N-terminal region of the hormone did not increase cyclic AMP formation, whereas they did stimulate increases in both DNA synthesis and CK activity. Moreover, the PTH fragment hPTH-(28-48) at 10 microM inhibited the increase in cyclic AMP caused by 10 nM-bPTH-(1-84). The increase of CK activity in ROS 17/2.8 cells caused by bPTH-(1-84) or hPTH-(28-48) was completely inhibited by either cycloheximide or actinomycin D, as was shown previously for rat calvaria cell cultures. These results indicated the presence of a functional domain of PTH in the central part of the molecule which exerts its mitogenic-related effects on osteoblast- and chondroblast-like cells in a cyclic AMP-independent manner. Since cyclic AMP formation by PTH leads to bone resorption, specific mid-region fragments of PTH might prove suitable for use in vivo to induce bone formation without concomitant resorption.     

Remodeling of the sagittal suture in osteopetrotic (op/op) mice associated with cranial flat bone growth.

It is well known that cranial flat bone experiences growth and development at the sutural interface, which is regarded as a neutral zone to control mechanical stimuli. In osteopetrotic (op/op) mice, meanwhile, cranial deformation is produced by the deficiency of osteoclasts and the subsequent defect of bone resorption. It would be a reasonable assumption that such disturbance in bone remodeling affects sutural modification and the relevant cranial flat bone development. The present study was thus conducted to examine histological features of the sagittal sutures in op/op mice, with special reference to the relevant bone remodeling. The sagittal sutures in 10-, 15-, 30-, and 60-day-old normal and op/op mice were observed microscopically. Furthermore, osteoclastic activity was evaluated on the sections stained with tartrate-resistant acid phosphatase (TRAP). The sutures of 15-day-old op/op mice showed stenosis and synostosis, and less-developed collagen fibers associated with an irregular arrangement of fibroblasts, whereas these changes were rarely found in normal mice. Osteoclasts were hardly detected in the parietal bones around the sutures of op/op mice, although the number was numerous in normal mice. These results emphasize that congenital deficiency in osteoclast produces unbalanced bone remodeling at the sutural interface and on the surfaces of the cranial bones, which is assumed to be closely related to cranial bone deformity in op/op mice.     

Amphiregulin is a novel growth factor involved in normal bone development and in the cellular response to parathyroid hormone stimulation

Parathyroid hormone (PTH) is the major mediator of calcium homeostasis and bone remodeling and is now known to be an effective drug for osteoporosis treatment. Yet the mechanisms responsible for its functions in bone are largely unknown. Here we report that the expression of amphiregulin (AR), a member of the epidermal growth factor (EGF) family, is rapidly and highly up-regulated by PTH in several osteoblastic cell lines and bone tissues. Other osteotropic hormones (1alpha,25-dihydroxyvitamin D3 and prostaglandin E2) also strongly stimulate AR expression. We found all EGF-like ligands and their receptors are expressed in osteoblasts, but AR is the only member that is highly regulated by PTH. Functional studies demonstrated that although AR is a potent growth factor for preosteoblasts, it completely inhibits further differentiation. AR also strongly and quickly stimulated Akt and ERK phosphorylation and c-fos and c-jun expression in an EGF receptor-dependent manner. Moreover, AR null mice displayed significantly less tibial trabecular bone than wild-type mice. Taken together, we have identified a novel growth factor that is PTH-regulated and appears to have an important role in bone metabolism.     

Impact of parathyroid hormone on bone marrow-derived stem cell mobilization and migration

Parathyroid hormone (PTH) is well-known as the principal regulator of calcium homeostasis in the human body and controls bone metabolism via actions on the survival and activation of osteoblasts. The intermittent administration of PTH has been shown to stimulate bone production in mice and men and therefore PTH administration has been recently approved for the treatment of osteoporosis. Besides to its physiological role in bone remodelling PTH has been demonstrated to influence and expand the bone marrow stem cell niche where hematopoietic stem cells, capable of both self-renewal and differentiation, reside. Moreover, intermittent PTH treatment is capable to induce mobilization of progenitor cells from the bone marrow into the bloodstream. This novel function of PTH on modulating the activity of the stem cell niche in the bone marrow as well as on mobilization and regeneration of bone marrow-derived stem cells offers new therapeutic options in bone marrow and stem cell transplantation as well as in the field of ischemic disorders.     

Incretin hormone receptors are required for normal beta cell development and function in female mice

The incretin hormones, glucose dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1), potentiate insulin secretion and are responsible for the majority of insulin secretion that occurs after a meal. They may also, however, have a fundamental role in pancreatic beta cell development and function, independently of their role in potentiating insulin secretion after a meal. This has led to observations that a loss of GIP or GLP-1 action affects normal beta cell function, however each one of the incretin hormones may compensate when the action of the other is lost and therefore the overall impact of the incretin hormones on beta cell function is not known. We therefore utilized a mouse line deficient in both the GLP-1 and GIP receptor genes, the double incretin receptor knockout (DIRKO), to determine the consequences of a lifelong, complete lack of incretin hormone action on beta cell function, in vivo, in intact animals. We found that DIRKO mice displayed impaired glucose tolerance and insulin secretion in response to both oral glucose and mixed meal tolerance tests compared to wild-type mice. Assessment of beta cell function using the hyperglycemic clamp technique revealed an 80% decrease in first phase insulin response in DIRKO mice, but a normal second phase insulin secretion. A similar decline was seen when wild-type mice were given acute intravenous injection of glucose together with the GLP-1 receptor antagonist Ex9-39. Ex vivo assessments of the pancreas revealed significantly fewer islets in the pancreata of DIRKO mice despite no differences in total pancreatic mass. Insulin secretion from isolated islets of DIRKO mice was impaired to a similar extent to that seen during the hyperglycemic clamp. Insulin secretion in wild-type islets was impaired by acute treatment with Ex9-39 to a similar extent as the in vivo intravenous glucose tolerance tests. In conclusion, a loss of the action of both incretin hormones results in direct impairment of beta cell function both in vivo and in vitro in a process that appears to be independent of the intestinally secreted incretin hormones. We therefore conclude that the incretin hormones together significantly impact both beta-cell function and beta-cell development.     

Midpalatal suture of osteopetrotic (op/op) mice exhibits immature fusion.

The midpalatal suture was observed histologically in both toothless osteopetrotic (op/op) and normal (control) mice. The normal mice had a mature sutural structure, which consists of a well-developed cartilage cell zone and palatal bone. In contrast, the thickness of the cartilage cell zone was substantially greater in the op/op mice than that in the controls. Moreover, the cartilage cells in the op/op mice were frequently found in the palatal bone as well as in the sutural space, exhibiting an imperfect fusion. It seems that immature fusion at the sutural interface in the op/op mice is related to a decrease in biting or masticatory force accompanied by the failure of tooth eruption in addition to an essential defect in osteoclast differentiation, which is a congenital symptom in op/op mice.     

Prostaglandin F(2alpha) negatively regulates bone resorption in murine osteoclast development

Prostaglandin (PG) E(2) promotes osteoclastic cell differentiation, but the physiological function of PGF(2alpha) remains unclear. We examined the physiological effects of PGF(2alpha) on osteoclast differentiation using a murine cell line, RAW264, and the column-purified murine bone marrow cells, both of which are differentiable into osteoclast-like multi-nuclear cells. Although PGF(2alpha) did not affect the number of differentiated osteoclasts, PGF(2alpha) reduced the bone resorption activity of osteoclasts developed from both cell types in a pit formation assay. Thus, PGF(2alpha) inhibits bone resorption without affecting the number of osteoclasts, providing a novel molecular mechanism underlying bone metabolism.     

Endogenous production of tumor necrosis factor by primary cultures of murine calvarial cells: influence on IL-6 production and osteoclast development

We have previously shown that tumor necrosis factor (TNF) and interleukin-1 (IL-1) acted synergistically to stimulate the production of IL-6 by bone marrow stromal and osteoblastic cells; and that an antibody to IL-6 inhibited TNF-induced osteoclast development in murine calvarial cell cultures. Prompted by this evidence, we have now examined whether TNF and/or IL-1 are produced by murine calvarial cells, and whether these cytokines are involved in IL-6 production and osteoclast formation. When cultured under basal conditions, calvarial cells produced TNF and IL-6, and were able to form bone resorbing osteoclasts. A neutralizing antibody against TNF suppressed both basal IL-6 production and the formation of bone resorbing osteoclasts. The anti-TNF antibody also inhibited IL-6 production in response to exogenous IL-1 or parathyroid hormone (PTH). In contrast, a neutralizing anti-IL-1 receptor antibody had no effect on basal, TNF- or PTH-stimulated IL-6 production. These findings suggest that TNF, but not IL-1, is produced by murine bone cells and that endogenous TNF induces the IL-6 production, osteoclast formation, and bone resorption exhibited by these cultures under basal conditions. Furthermore, bone cell-derived TNF amplifies the stimulatory effect of exogenous IL-1 or PTH on IL-6 production by calvarial cells.