Itai Bab

Hereditary Hypophosphatemic Rickets with Hypercalciuria

We studied a new hereditary syndrome of hypophosphatemic rickets and hypercalciuria in six affected members of one kindred. In all patients, the manifestations of disease began in early childhood. The characteristic features are rickets, short stature, increased renal phosphate clearance (the ratio between the maximal tubular reabsorption rate for phosphorus and the glomerular filtration rate [TmP/GFR] is 2 to 4 S.D. below the age-related mean), hypercalciuria (8.6 mg of urinary calcium per kilogram of body weight per 24 hours vs. the upper normal value of 4.0), normal serum calcium levels, increased gastrointestinal absorption of calcium and phosphorus, an elevated serum concentration of 1,25-dihydroxyvitamin D (390 +/- 99 pg per milliliter vs. the upper normal value of 110), and suppressed parathyroid function (an immunoreactive parathyroid hormone level of 0.33 +/- 0.1 ng per milliliter and a cyclic AMP level of 1.39 +/- 0.12 nmol per deciliter of glomerular filtrate vs. the lower normal values of 0.3 and 1.5, respectively). Long-term phosphate supplementation as the sole therapy resulted in reversal of all clinical and biochemical abnormalities except the decreased TmP/GFR. We propose that the pivotal defect in this syndrome is a renal phosphate leak resulting in hypophosphatemia with an appropriate elevation of 1,25-dihydroxyvitamin D levels, which causes increased calcium absorption, parathyroid suppression, and hypercalciuria. This syndrome may represent one end of a spectrum of hereditary absorptive hypercalciuria. Our observations support the importance of phosphate as a mediator in controlling 1,25-dihydroxyvitamin D production in human beings.

Human Parathyroid Hormone 1–34 Reverses Bone Loss in Ovariectomized Mice

The experimental work characterizing the anabolic effect of parathyroid hormone (PTH) in bone has been performed in nonmurine ovariectomized (OVX) animals, mainly rats. A major drawback of these animal models is their inaccessibility to genetic manipulations such as gene knockout and overexpression. Therefore, this study on PTH anabolic activity was carried out in OVX mice that can be manipulated genetically in future studies. Adult Swiss‐Webster mice were OVX, and after the fifth postoperative week were treated intermittently with human PTH(1–34) [hPTH(1–34)] or vehicle for 4 weeks. Femoral bones were evaluated by microcomputed tomography (μCT) followed by histomorphometry. A tight correlation was observed between trabecular density (BV/TV) determinations made by both methods. The BV/TV showed >60% loss in the distal metaphysis in 5‐week and 9‐week post‐OVX, non‐PTH‐treated animals. PTH induced a ∼35% recovery of this loss and a ∼40% reversal of the associated decreases in trabecular number (Tb.N) and connectivity. PTH also caused a shift from single to double calcein‐labeled trabecular surfaces, a significant enhancement in the mineralizing perimeter and a respective 2‐ and 3‐fold stimulation of the mineral appositional rate (MAR) and bone formation rate (BFR). Diaphyseal endosteal cortical MAR and thickness also were increased with a high correlation between these parameters. These data show that OVX osteoporotic mice respond to PTH by increased osteoblast activity and the consequent restoration of trabecular network. The Swiss‐Webster mouse model will be useful in future studies investigating molecular mechanisms involved in the pathogenesis and treatment of osteoporosis, including the mechanisms of action of known and future bone antiresorptive and anabolic agents.

Endocannabinoid signaling at the periphery: 50 years after THC

In 1964, the psychoactive ingredient of Cannabis sativa, Δ(9)-tetrahydrocannabinol (THC), was isolated. Nearly 30 years later the endogenous counterparts of THC, collectively termed endocannabinoids (eCBs), were discovered: N-arachidonoylethanolamine (anandamide) (AEA) in 1992 and 2-arachidonoylglycerol (2-AG) in 1995. Since then, considerable research has shed light on the impact of eCBs on human health and disease, identifying an ensemble of proteins that bind, synthesize, and degrade them and that together form the eCB system (ECS). eCBs control basic biological processes including cell choice between survival and death and progenitor/stem cell proliferation and differentiation. Unsurprisingly, in the past two decades eCBs have been recognized as key mediators of several aspects of human pathophysiology and thus have emerged to be among the most widespread and versatile signaling molecules ever discovered. Here some of the pioneers of this research field review the state of the art of critical eCB functions in peripheral organs. Our community effort is aimed at establishing consensus views on the relevance of the peripheral ECS for human health and disease pathogenesis, as well as highlighting emerging challenges and therapeutic hopes.

Oxytocin is an anabolic bone hormone

We report that oxytocin (OT), a primitive neurohypophyseal hormone, hitherto thought solely to modulate lactation and social bonding, is a direct regulator of bone mass. Deletion of OT or the OT receptor (Oxtr) in male or female mice causes osteoporosis resulting from reduced bone formation. Consistent with low bone formation, OT stimulates the differentiation of osteoblasts to a mineralizing phenotype by causing the up-regulation of BMP-2, which in turn controls Schnurri-2 and 3, Osterix, and ATF-4 expression. In contrast, OT has dual effects on the osteoclast. It stimulates osteoclast formation both directly, by activating NF-κB and MAP kinase signaling, and indirectly through the up-regulation of RANK-L. On the other hand, OT inhibits bone resorption by mature osteoclasts by triggering cytosolic Ca2+ release and NO synthesis. Together, the complementary genetic and pharmacologic approaches reveal OT as a novel anabolic regulator of bone mass, with potential implications for osteoporosis therapy.

Depression induces bone loss through stimulation of the sympathetic nervous system

Major depression is associated with low bone mass and increased incidence of osteoporotic fractures. However, causality between depression and bone loss has not been established. Here, we show that mice subjected to chronic mild stress (CMS), an established model of depression in rodents, display behavioral depression accompanied by impaired bone mass and structure, as portrayed by decreases in trabecular bone volume density, trabecular number, and trabecular connectivity density assessed in the distal femoral metaphysis and L3 vertebral body. Bone remodeling analysis revealed that the CMS-induced skeletal deficiency is accompanied by restrained bone formation resulting from reduced osteoblast number. Antidepressant therapy, which prevents the behavioral responses to CMS, completely inhibits the decrease in bone formation and markedly attenuates the CMS-induced bone loss. The depression-triggered bone loss is associated with a substantial increase in bone norepinephrine levels and can be blocked by the β-adrenergic antagonist propranolol, suggesting that the sympathetic nervous system mediates the skeletal effects of stress-induced depression. These results define a linkage among depression, excessive adrenergic activity, and reduced bone formation, thus demonstrating an interaction among behavioral responses, the brain, and the skeleton, which leads to impaired bone structure. Together with the common occurrence of depression and bone loss in the aging population, the present data implicate depression as a potential major risk factor for osteoporosis and the associated increase in fracture incidence.

Histone H4-related osteogenic growth peptide (OGP): a novel circulating stimulator of osteoblastic activity.

It has been established that regenerating marrow induces an osteogenic response in distant skeletal sites and that this activity is mediated by factors released into the circulation by the healing tissue. In the present study we have characterized one of these factors, a 14 amino acid peptide named osteogenic growth peptide (OGP). Synthetic OGP, identical in structure to the native molecule, stimulates the proliferation and alkaline phosphatase activity of osteoblastic cells in vitro and increases bone mass in rats when injected in vivo. Immunoreactive OGP in high abundance is present physiologically in the serum, mainly in the form of an OGP‐OGP binding protein complex. A marked increase in serum bound and unbound OGP accompanies the osteogenic phase of post‐ablation marrow regeneration and associated systemic osteogenic response. Authentic OGP is identical to the C‐terminus of histone H4 and shares a five residue motif with a T‐cell receptor beta‐chain V‐region and the Bacillus subtilis outB locus. Since these latter proteins have not been implicated previously in the control of cell proliferation or differentiation, OGP may belong to a novel, heretofore unrecognized family of regulatory peptides. Perhaps more importantly, OGP appears to represent a new class of molecules involved in the systemic control of osteoblast proliferation and differentiation.

The cannabinoid CB1 receptor regulates bone formation by modulating adrenergic signaling

We have recently reported that in bone the cannabinoid CB1 receptor is present in sympathetic terminals. Here we show that traumatic brain injury (TBI), which in humans enhances peripheral osteogenesis and fracture healing, acutely stimulates bone formation in a distant skeletal site. At this site we demonstrate i) a high level of the main endocannabinoid, 2‐arachidonoylglycerol (2‐AG), and expression of diacylglycerol lipases, enzymes essential for 2‐AG synthesis;ii) that the TBI‐induced increase in bone formation is preceded by elevation of the 2‐AG and a decrease in norepinephrine (NE) levels. The TBI stimulation of bone formation was absent in CB1‐null mice. In wild‐type animals it could be mimicked, including the suppression of NE levels, by 2‐AG administration. The TBI‐ and 2‐AG‐induced stimulation of osteogenesis was restrained by the β‐adrenergic receptor agonist isoproterenol. NE from sympathetic terminals is known to tonically inhibit bone formation by activating osteoblastic β2‐adrenergic receptors. The present findings further demonstrate that the sympathetic control of bone formation is regulated through 2‐AG activation of prejunctional CB1. Elevation of bone 2‐AG apparently suppresses NE release from bone sympathetic terminals, thus alleviating the inhibition of bone formation. The involvement of osteoblastic CB2 signaling in this process is minimal, if any.— Tam, J., Trembovler, V., Di Marzo, V., Petrosino, S., Leo, G., Alexandrovich, A., Regev, E., Casap, N., Shteyer, A., Ledent, C., Karsak, M., Zimmer, A., Mechoulam, R., Yirmiya, R., Shohami, E., Bab, I. The cannabinoid CB1 receptor regulates bone formation by modulating adrenergic signaling. FASEB J. 22, 285–294 (2008)

Involvement of neuronal cannabinoid receptor CB1 in regulation of bone mass and bone remodeling.

The CB1 cannabinoid receptor has been implicated in the regulation of bone remodeling and bone mass. A high bone mass (HBM) phenotype was reported in CB1-null mice generated on a CD1 background (CD1CB1-/- mice). By contrast, our preliminary studies in cb1-/- mice, backcrossed to C57BL/6J mice (C57CB1-/- mice), revealed low bone mass (LBM). We therefore analyzed CB1 expression in bone and compared the skeletons of sexually mature C57CB1-/- and CD1CB1-/- mice in the same experimental setting. CB1 mRNA is weakly expressed in osteoclasts and immunoreactive CB1 is present in sympathetic neurons, close to osteoblasts. In addition to their LBM, male and female C57CB1-/- mice exhibit decreased bone formation rate and increased osteoclast number. The skeletal phenotype of the CD1CB1-/- mice shows a gender disparity. Female mice have normal trabecular bone with a slight cortical expansion, whereas male CD1CB1-/- animals display an HBM phenotype. We were surprised to find that bone formation and resorption are within normal limits. These findings, at least the consistent set of data obtained in the C57CB1-/- line, suggest an important role for CB1 signaling in the regulation of bone remodeling and bone mass. Because sympathetic CB1 signaling inhibits norepinephrine (NE) release in peripheral tissues, part of the endocannabinoid activity in bone may be attributed to the regulation of NE release from sympathetic nerve fibers. Several phenotypic discrepancies have been reported between C57CB1-/- and CD1CB1-/- mice that could result from genetic differences between the background strains. Unraveling these differences can provide useful information on the physiologic functional milieu of CB1 in bone.

Cannabinoid receptors and the regulation of bone mass

A functional endocannabinoid system is present in several mammalian organs and tissues. Recently, endocannabinoids and their receptors have been reported in the skeleton. Osteoblasts, the bone forming cells, and osteoclasts, the bone resorbing cells, produce the endocannabinoids anandamide and 2‐arachidonoylglycerol and express CB2 cannabinoid receptors. Although CB2 has been implicated in pathological processes in the central nervous system and peripheral tissues, the skeleton appears as the main system physiologically regulated by CB2. CB2‐deficient mice show a markedly accelerated age‐related bone loss and the CNR2 gene (encoding CB2) in women is associated with low bone mineral density. The activation of CB2 attenuates ovariectomy‐induced bone loss in mice by restraining bone resorption and enhancing bone formation. Hence synthetic CB2 ligands, which are stable and orally available, provide a basis for developing novel anti‐osteoporotic therapies. Activation of CB1 in sympathetic nerve terminals in bone inhibits norepinephrine release, thus balancing the tonic sympathetic restrain of bone formation. Low levels of CB1 were also reported in osteoclasts. CB1‐null mice display a skeletal phenotype that is dependent on the mouse strain, gender and specific mutation of the CB1 encoding gene, CNR1.

Intermittent recombinant TSH injections prevent ovariectomy-induced bone loss

We recently described the direct effects of thyroid-stimulating hormone (TSH) on bone and suggested that the bone loss in hyperthyroidism, hitherto attributed solely to elevated thyroid hormone levels, could at least in part arise from accompanying decrements in serum TSH. Recent studies on both mice and human subjects provide compelling evidence that thyroid hormones and TSH have the opposite effects on the skeleton. Here, we show that TSH, when injected intermittently into rodents, even at intervals of 2 weeks, displays a powerful antiresorptive action in vivo. By virtue of this action, together with the possible anabolic effects shown earlier, TSH both prevents bone loss and restores the lost bone after ovariectomy. Importantly, the osteoclast inhibitory action of TSH persists ex vivo even after therapy is stopped for 4 weeks. This profound and lasting antiresorptive action of TSH is mimicked in cells that genetically overexpress the constitutively active ligand-independent TSH receptor (TSHR). In contrast, loss of function of a mutant TSHR (Pro → Leu at 556) in congenital hypothyroid mice activates osteoclast differentiation, confirming once again our premise that TSHRs have a critical role in regulating bone remodeling.