bone is a dynamic tissue which undergoes continuous self - renewal , and bone homeostasis relies on functional equilibrium among three types of cells : osteoclasts essential for bone resorption , osteoblasts responsible for bone matrix formation , and osteocytes involved in the reception and transduction of mechanical stimuli and in the regulation of osteoclast / osteoblast differentiation and function .
the balance between bone synthesis and resorption is finely tuned and any perturbations of this balance in adults trigger bone disease .
osteopetrosis ( osteo : bone and petros : stone ) regroups a set of rare , heterogeneous , and inherited bone diseases characterized by increased bone mass .
osteopetrosis is therefore an osteocondensing disease . in principle , two causes could give rise to this osteocondensing phenotype : increased bone formation or failure of resorption by osteoclasts .
however , osteopetrosis is known to result from defective osteoclast differentiation or function [ 3 , 4 ] .
important progress has been made during the past decades in understanding the molecular mechanisms underlying the development of hereditary diseases characterized by increased bone mass [ 3 , 5 ] .
our objective in this review is not to give a detailed description of all the sclerosing bone diseases ; such information can be found in other reviews [ 3 , 4 , 6 , 7 ] .
instead , we discuss recent findings regarding osteopetrosis and how the study of this disease has contributed to new understanding of functions associated with the skeleton [ 810 ] .
osteoclasts are highly specialized cells responsible for the dissolution of bone mineral and for the degradation of organic matrix .
osteoclasts are multinucleated cells ( containing up to 50 nuclei ) , derived from the fusion of mononuclear cells belonging to the monocyte - macrophage lineage . under the influence of factors secreted by osteoblasts and/or stromal cells present in the bone microenvironment
m - csf ( macrophage colony stimulating factor ) is expressed by osteoblasts and binds the c - fms receptor on osteoclast precursors , stimulating their proliferation and the expression of rank ( receptor activator of nf-b ) receptor .
the interaction of rank - l , expressed and secreted by osteoblasts and stromal cells , with its receptor propels the fusion of osteoclast progenitors to form a giant multinucleated cell .
a terminally differentiated osteoclast is able to degrade extracellular bone matrix by the action of specific proteins . to resorb bone matrix , osteoclasts must be perfectly polarized with a ruffle border and a sealing zone .
these two features allow the creation of a resorption lacuna into which h ions are actively secreted in order to acidify it , leading to dissolution of bone matrix hydroxyapatite .
type ii carbonic anhydrase ( caii ) hydrates co2 to form carbonic acid , which spontaneously dissociates into protons and hco3 ions .
the protons are actively transported into the resorption lacuna by a vacuolar atpase proton pump located in the ruffled border domain [ 1 , 11 ] .
the hco3 ion is exchanged with cl by a bicarbonate / chloride antiport on the basolateral membrane of the cell .
the chloride ion is translocated into the resorption lacuna through chloride channel 7 ( clcn7 ) , recently reclassified as chloride / proton antiport .
the acidic environment promotes the dissolution of inorganic content and also exposes the organic matrix , which is then ready to be digested by secreted proteolytic enzymes [ 1 , 11 ] .
the collagenous bone matrix is dissolved by two groups of enzymes , the matrix metalloproteases and the lysosomal cathepsins .
this enzyme is secreted into the resorption lacuna and degrades type i collagen in the acidic environment .
the acquisition and maintenance of osteoclast membrane polarity require a complex system of vesicle trafficking and ongoing cytoskeletal renewal .
one of the proteins involved in these processes is plekhm1 ( pleckstrin homolog domain containing family m with run domain member 1 ) .
this protein plays a crucial role in acidification and trafficking of intracellular vesicles [ 13 , 14 ] . a recently discovered protein important for osteoclast trafficking activity is snx10 ( sorting nexin 10 ) .
snx10 belongs to a family of about 30 proteins sharing the px ( phox homology ) phospholipid binding domain and is involved in protein trafficking and osteoclast differentiation / function [ 15 , 16 ] .
osteopetrosis is a generic name for a group of rare genetic bone diseases characterized by osteoclast failure .
the adult autosomal dominant type ii form or albert - schnberg disease classified as mild is sometimes associated with bone symptoms .
in contrast , the infantile recessive osteopetroses are severe forms and usually lethal in childhood without treatment [ 3 , 57 , 17 ] .
mutations in at least 8 genes ( table 1 ) have been identified as being responsible for osteopetrosis pathogenesis in humans .
autosomal recessive osteopetrosis is a severe disease diagnosed in the first months of life owing to a variety of problems .
they also show bruising and frequent bleeding secondary to medullar hyperplasia caused by bony invasion of the medullar space .
untreated children usually die during their decade from hemorrhage , pneumonia , anemia , or infection .
hematopoietic stem cell transplantation ( hsct ) is the only treatment option currently far available [ 3 , 5 , 18 ] .
the number of osteoclasts is normal or high , but their acidifying activity , compulsory for bone resorption , is impaired .
several genes are known to be involved in this form of osteopetrosis ( table 1 , figure 1 ) . about 50% of aro patients harbors loss - of - function mutations of tcirg1 which codes for the proton pump v - atpase 3 subunit [ 17 , 19 ] .
loss - of - function mutations of the clcn7 gene , coding for chloride channel 7 , have also been described in ~10% of aro patients .
mutations in ostm1 ( osteopetrosis associated transmembrane protein 1 ) , coding for a protein involved in transport of clcn7 to the ruffled border ( and considered as a  subunit of clcn7 ) , have been described as causing severe osteopetrosis in ~5% of patients [ 2123 ] .
primary neurological defects can also be present in patients bearing ostm1 or clcn7 mutations [ 20 , 23 ] .
deformity of the distal femora , bone pain , and chondrolysis of the left hip were described in one patient .
recently , a mutation in the snx10 gene was found in 15 families in which the patients displayed a heterogeneous phenotype .
mild growth retardation , hypocalcemia , hydrocephalus , severe hematological abnormalities , and visual impairment have been described in patients with loss of function mutations of snx10 [ 3 , 15 , 16 , 24 ] .
less than 4% of aro patients harbors loss - of - function mutations of tnfsf11 , encoding rank - l , or of tnfrsf11a , encoding rank receptor , and constitute a distinct subgroup of recessive osteopetrosis .
indeed , bone biopsies from these patients revealed a complete lack of osteoclasts [ 2527 ] .
in addition , patients with tnfsf11 mutations exhibit some immune abnormalities and not palpable lymph nodes , but b and t lymphocyte numbers are normal .
by contrast , most of the patients with tnfrsf11a mutations have a more severe immunological phenotype with a defect in memory b lymphocyte differentiation and a reduction in immunoglobulins levels [ 2527 ] .
however , osteopetrosis caused by tnfsf11 mutations can not be treated by hsct , because an osteoblast defect is the basis of this pathology . in practice ,
a molecular genetic diagnosis should be made before transplantation to ensure that the pathology is not due to a rank - l mutation . in 1983 ,
renal tubular acidosis can explain the hypotonia , apathy , and muscular weakness occurrence in some patients .
by radiography caii deficiency resembles other forms of osteopetrosis , but brain calcifications can develop during childhood and osteosclerosis and bone modeling spontaneously decrease instead of increasing in the course of pathology evolution .
metabolic acidosis occurs during the neonatal period , and renal tubular acidosis , both proximal and distal , has been described [ 29 , 30 ] .
caii is expressed in many different tissues including brain , kidney , red blood cells , cartilage , lung , and digestive mucosa .
ado ii is commonly called benign osteopetrosis but presents with an extremely heterogeneous course from asymptomatic to rarely fatal .
adoii clinical and radiological signs occur quite late in childhood or in the teens , although earlier occurring has sometimes been reported .
adoii patients usually displayed osteosclerosis at the vertebral level ( so - called sandwich vertebrae ) and also a bone in bone aspect observed mainly in the iliac bones , but sometimes in other epiphyses .
in addition , on radiography , alternating dense and light bands are often seen in iliac bones and at the extremities of long bones [ 7 , 33 ] .
bone fractures occur in 80% of patients , with a mean of 3 fractures per patient .
the femur is the most fractured bone in this pathology , but fractures can occur on any long bones and even at the posterior arch of the vertebrae , which often leads to a spondylolisthesis .
hip arthritis is frequent ( in 50% of the cases ) and could be due to excessive stiffness of the subchondral bone .
auditory or visual impairment occurs in less than 5% of affected individuals [ 7 , 33 ] .
arthropathies are technically difficult and postsurgical complications , such as strengthening delay , infections , and pseudoarthritis are frequent ( 50% of cases ) due to bone stiffness .
for example , in 3 families in which most of the affected individuals expressed only a mild form of adoii , some members exhibited anemia and blindness caused by optical nerve compression .
this phenotype has been called intermediate osteopetrosis because of its overlap with that of mild aro . about 70% of patients affected by adoii harbors heterozygous dominant negative mutations of the clcn7 gene ( figure 1 , table 1 ) .
in the remaining ~30% of cases , no mutations in clcn7 gene sequences were found , suggesting involvement of further genes in the pathogenesis of this form of osteopetrosis .
osteopetrorickets is a bone disorder characterized by increase of bone mass with a defect of skeletal mineralization .
schinke and coauthors performed histological analysis of undecalcified bone biopsies of 21 patients who received a diagnosis of osteopetrosis . in patients with loss - of - function mutations in the tcirg1 gene ,
the same pathological enrichment of osteoid was confirmed in oc / oc mice carrying a loss - of - function mutation of the tcirg1 gene , while no increase was revealed in osteopetrotic scr mice .
the increase of osteoid volume was associated with hypocalcemia , due to a defect of intestinal calcium uptake .
indeed it was shown that tcirg1 is also expressed in the fundus , a region of the stomach involved in gastric acidification , and loss - of - function mutations induce hypochlorhydria and reduced intestinal calcium uptake in both humans and mice .
gastric acidification is a prerequisite for efficient intestinal calcium uptake ; in hypochlorhydria , intestinal calcium uptake is lowered leading to parathyroin hormone ( pth)-dependent activation of osteoclasts and an osteoporosis phenotype . in the case of loss - of - function mutation of tcirg1 ,
intestinal calcium uptake is reduced and pth - dependent stimulation of bone resorption is blocked , resulting in an osteopetrorickets phenotype .
performed histomorphometric analysis of bone biopsies of 9 osteopetrotic patients with loss - of - mutation in the tcirg1 , clcn7 , and tnfsf11a genes .
in contrast , there was no sign of osteopetrorickets in patients with clcn7 and tnfsf11a gene mutations [ 3537 ] .
osteocalcin can exist in two different forms , undercarboxylated and carboxylated on 3 glutamic acid residues [ 10 , 38 ] .
the carboxylated form has high affinity for the hydroxyapatite , facilitating its engraftment in the bone matrix .
ferron et al . investigated whether acidic bone resorption lacuna promotes the decarboxylation of osteocalcin .
indeed they observed that in oc / oc mice the levels of undercarboxylated osteocalcin were reduced by 30% compared to wild - type animals .
similar features were observed in wild - type mice that received fetal liver hematopoietic stem cells ( hscs ) from oc / oc mice confirming the relevance of osteoclast function in osteocalcin - insulin signaling .
moreover they observed that oc / oc mice were glucose intolerant , with reduced serum insulin levels , pancreas insulin content , and insulinexpression in the pancreas [ 40 , 41 ] .
interestingly , it was shown that osteopetrotic patients affected by autosomal dominant osteopetrosis with osteoclast acidification defects have lower levels of insulin and a lower undercarboxylated / carboxylated osteocalcin ratio but diabetes was not reported [ 40 , 41 ] .
osteocalcin is very important for the cross talk between bone and the systems responsible for male fertility [ 42 , 43 ] .
karsenty 's group showed that osteocalcin is able to stimulate , in a camp response element binding ( creb ) protein - dependent manner , the production of testosterone by testes .
this function is mediated by the interaction of osteocalcin with gprc6a , a g - coupled receptor expressed in leydig cells [ 42 , 43 ] . in 1997 cohen et al
.   showed that op / op mice ( which lack colony stimulating factor 1 , csf-1 ) have reduced mating ability , low sperm numbers , and low serum testosterone levels due to decreased leydig cell steroidogenesis .
the study also showed how csf-1 is essential for the development and function of the hypothalamic - pituitary - gonadal axis .
further studies in osteopetrotic animal models will be important to confirm the interaction between bone and male fertility .
it now well established that there is a tight correlation between bone and the immune system , which has led to a new discipline called osteoimmunology .
this research area is just now expanding and we are beginning to better understand the relevance of this interplay in bone diseases .
associated defects in b cell function were attributed to mutations in genes involved in osteoclast differentiation or function or to an abnormal medullary microenvironment .
oc / oc mice display a block at the pro - b to pre - b cell transition , which is due to a defect of the bone microenvironment rather than to a cell autonomous defect of b cells , because in vitro experiments showed that b cell progenitors isolated from osteopetrotic mice were able to differentiate into immature b cells
apart the alterations of b cell differentiation , kong and coauthors described a reduction in thymus size and a block of thymocyte development at the cd4cd8cd44cd25 stage .
many studies have been published regarding the effects on osteoclast differentiation and function following alterations of immune cells .
in particular , it was shown that animal overexpressing the transcription factor foxp3 ( forkhead box p3 ) displayed osteopetrotic phenotype with increased bone mass and reduced osteoclast number and activity . moreover in vitro experiments suggested that treg cells could inhibit osteoclast differentiation and function by suppression of cytoskeletal reorganization .
bone and bone marrow can be considered as two distinct compartments of the same functional unit , the bone - bone marrow organ .
indeed it was shown that dysfunction of osteoclast activity results in aberrant formation of the hsc niche , leading to retention of hsc in the spleen .
the frequency and absolute number of linnegsca1ckit ( lsk cells ) were decreased by 90% and 99.8% , respectively , in the bone marrow of oc / oc mice compared to controls .
this alteration was associated with a defect of mesenchymal stem cells to differentiate into osteoblasts .
the effect was revealed by a dramatic reduction in the expression of the osteoblast markers runx2 , alp , osteocalcin and bsp and a reduced proportion of cells expressing cd51 and the integrin 5 ( cd49e ) .
the study showed that osteoclasts promote the formation of the hsc niche , regulating the osteoblast differentiation important for the niche .
indeed the authors showed that the absence of osteoclast activity affects formation of the bone marrow hsc niche and impairs ability of mesenchymal stem / stromal cells to recruit hematopoietic progenitor cells .
moreover the restoration of osteoclast function by treatment with cd45sca1 cells reestablishes normal levels of hematopoietic progenitors in the bone marrow [ 46 , 53 ] .
the relationship between bone and adipose tissue is an area of intensive investigations because molecules involved in bone - fat interactions could be used as pharmacological targets to prevent osteoporosis and bone fractures . in particular ,
ligands for ppar- include long - chain fatty acid and synthetic compounds such as thiazolidinedione .
ppar- functions are associated with activation of the adipogenesis and inhibition of the osteoblastogenesis [ 56 , 57 ] .
these mice developed increased bone mass with a parallel reduction of bone marrow cavities and extramedullary hematopoiesis .
indeed deletion of ppar- resulted in impaired osteoclast differentiation and activity , since it regulates c - fos expression involved in rankl signaling .
demonstrated that the absence of ppar- in white adipose tissue led to lipodystrophy , increased bone mineral density , and extramedullary hematopoiesis in spleen .
this interplay between bone and adipose tissue has clinical important implications , since a long - term treatment with the ppar- agonist rosiglitazone in patients affected by type 2 diabetes could result in osteoporosis and bone fractures [ 54 , 58 ] .
rare hereditary diseases inducing a bone condensation have shed new light on several aspects of bone cellular biology that were not well known .
indeed the study of these diseases allowed the identification of new mechanisms of osteoclast differentiation and function and the discovery of new functions associated with the skeleton .
much evidence suggests that the skeleton has a central role in bone physiology since bone disorders usually impact other organs [ 8 , 9 , 42 , 43 , 45 , 53 , 54 , 60 ] .
however , there are some features of these diseases that require further investigation . for example , as in other monogenic diseases , the genotype - phenotype correlation is not always clear and consistent . indeed , the same mutations can give rise to different phenotypes , as exemplified by the clcn7 gene heterozygous mutations .
efforts to identify the mutations responsible for the remaining 30% are on - going . from a pathophysiological point of view , it is worth noting that the pathologies caused by reduced osteoclastic activity such as osteopetrosis lead to frequent fractures .
this might be linked to a skeleton elasticity defect , but also to an inability to repair micro damage in bones because of a lower rate of bone turnover .
this situation illustrates the well - known discrepancy between the bone quantity and its resistance to mechanical stress .
in contrast , pathologies caused by an increase in bone formation due to increased activity of the wnt signaling pathway ( striated osteopathy ) or to tgf activating mutations ( camurati engelmann disease ) are not associated with an increased incidence of fractures . in conclusion ,
further study of osteopetrosis will allow us to better understand the physiology of bone and its impact on the whole body
. moreover our challenge for the future will be to identify new therapeutic approaches for this disabling disease , particularly for those forms for which only palliative intervention is currently available .
 osteopetrosis is a rare genetic disorder characterized by an increase of bone mass due to defective osteoclast function . 
 patients typically displayed spontaneous fractures , anemia , and in the most severe forms hepatosplenomegaly and compression of cranial facial nerves leading to deafness and blindness . 
 osteopetrosis comprises a heterogeneous group of diseases as several forms are known with different models of inheritance and severity from asymptomatic to lethal . 
 this review summarizes the genetic and clinical features of osteopetrosis , emphasizing how recent studies of this disease have contributed to understanding the central role of the skeleton in the whole body physiology . 
 in particular , the interplay of bone with the stomach , insulin metabolism , male fertility , the immune system , bone marrow , and fat is described . 