Barbara Peruzzi

Involvement of PLEKHM1 in osteoclastic vesicular transport and osteopetrosis in incisors absent rats and humans

This study illustrates that Plekhm1 is an essential protein for bone resorption, as loss-of-function mutations were found to underlie the osteopetrotic phenotype of the incisors absent rat as well as an intermediate type of human osteopetrosis. Electron and confocal microscopic analysis demonstrated that monocytes from a patient homozygous for the mutation differentiated into osteoclasts normally, but when cultured on dentine discs, the osteoclasts failed to form ruffled borders and showed little evidence of bone resorption. The presence of both RUN and pleckstrin homology domains suggests that Plekhm1 may be linked to small GTPase signaling. We found that Plekhm1 colocalized with Rab7 to late endosomal/lysosomal vesicles in HEK293 and osteoclast-like cells, an effect that was dependent on the prenylation of Rab7. In conclusion, we believe PLEKHM1 to be a novel gene implicated in the development of osteopetrosis, with a putative critical function in vesicular transport in the osteoclast.

A new heterozygous mutation (R714C) of the osteopetrosis gene, pleckstrin homolog domain containing family M (with run domain) member 1 (PLEKHM1), impairs vesicular acidification and increases TRACP secretion in osteoclasts.

We studied phenotypic and cellular aspects in a patient with a heterozygous mutation of the PLEKHM1 gene and obtained some indications regarding the role of the protein in bone cell function. Plekhm1 is involved in osteoclast endosomal vesicle acidification and TRACP exocytosis, contributing to events involved in osteoclast–osteoblast cross‐talk.

c-Src and IL-6 inhibit osteoblast differentiation and integrate IGFBP5 signalling

Interleukin-6 (IL-6) and c-Src impair osteoblast maturation in vitro and in vivo. Given the similar effects of these factors, they are likely to establish a functional loop to maintain osteoblasts in a less mature status. Here we describe a pathway whereby c-Src stimulates IL-6 expression through the STAT3 factor, which, in response to IL-6 induces insulin-like growth factor 5 (IGFBP5), a c-Src activating factor that amplifies this loop only in immature osteoblasts. In contrast, in mature osteoblasts, IGFBP5 is enhanced by Runx2, but is no longer able to stimulate c-Src activation, as this tyrosine kinase at this stage is downregulated. We find that the IGFBP5 produced by osteoblasts stimulates osteoclastogenesis and bone resorption, acting as an osteoblast-osteoclast coupling factor. Finally, we demonstrate that the integrated actions of c-Src, IL-6 and IGFBP5 also have a role in vivo. We conclude that this pathway is relevant for bone metabolism, both in physiological and in pathological conditions.

The Physiology and Pathophysiology of the Osteoclast

The osteoclast is the giant cell of bone phylogenically evolved to resorb the bone matrix. It contributes to both the bone modeling and remodeling processes through a complex extracellular mechanism, termed bone resorption, which removes the mineral and the organic bone matrix components. Primary osteoclast pathologies occur both in children and adults, and are very common in elderly. Reduced bone resorption is typically responsible of osteopetrosis, in which both bone modeling and remodeling are altered, and the primary bone persists reducing strength and causing spontaneous fractures, failure in hematopoiesis and impairment of sensory and motor systems. In children, bone resorption can be increased secondary to other pathological conditions due for instance to mechanical failure or inflammatory diseases. Excess bone resorption over formation is typical of post-menopausal women and elderly men, and the net result of this unbalanced bone cell activity is osteoporosis, which affects millions of people worldwide with high costs for management. This article will expand the concepts associated with the pathophysiology of the osteoclasts, providing up-to-date information on their function and involvement in bone diseases.

Committed Osteoclast Precursors Colonize the Bone and Improve the Phenotype of a Mouse Model of Autosomal Recessive Osteopetrosis

Osteopetrosis is a genetic disease characterized by defective osteoclasts. Autosomal recessive osteopetrosis is fatal within the first years of life. Hematopoietic stem cell transplantation (HSCT) cures fewer than 50% of cases but often leaves severe neurologic damages and other dysfunctions. Osteoclast appearance after HSCT is a slow process, during which disease progression continues. We hypothesize that a support osteoclast precursor therapy may contribute to improve the osteopetrotic phenotype. To this end, we established a procedure to obtain the best yield of osteoclast precursors from human peripheral blood or mouse bone marrow mononuclear cells. These cells were injected in vivo in animal models, testing different cell injection protocols, as well as in association with CD117+ stem cells. Injected cells showed the ability to form multinucleated osteoclasts and to improve the phenotype of oc/oc osteopetrotic mice. In the best working protocol, animals presented with longer survival, improved weight and longitudinal growth, increased tibial length, tooth eruption, decreased bone volume, reduced bone marrow fibrosis, and improved hematopoiesis compared with sham‐treated mice. These results provide first‐hand information on the feasibility of a support osteoclast precursor therapy in osteopetrosis.

The Crucial Role of c-Src Tyrosine Kinase in Bone Metabolism

Due to its proto-oncogene nature, c-Src is the SFK most frequently associated with malignancy (Yeatman, 2004). Over 100 years ago, Peyton Rous observed that injection of cell-free extracts from tumours grown in chickens caused the development of the same type of tumour in host animals. This observation prompted the hypothesis that a filterable agent was the cause of the tumour (Rous, 1911a, 1911b). In support of this notion, in 1955 Rubin showed that the Rous’filterable agent was a virus, called Rous Sarcoma Virus (RSV), which was found to play a direct role in inducing cell malignancy (Rubin, 1955). In the ‘60s and ‘70s, the tools of modern molecular biology provided the genetic definition of v-Src, a viral oncogene included within the RSV genome. v-Src was observed not to be required for virus replication but to be the causative agent of cancer (Martin, 1970; Duesberg & Vogt, 1970). Shortly thereafter, it was shown that v-Src had a counterpart in eukaryotic cells, named c-Src (Takeda and Hanafusa, 1983). c-Src is involved in many physiological functions of the cells. It carries a regulatory domain lacking in v-Src (Fig. 1), therefore, its activity is under tight molecular control. c-Src was the first of several proto-oncogenes discovered in the vertebrate genome and, in 1989, this discovery earned Bishop and Varmus the Nobel Prize in Physiology or Medicine, for the description of “the cellular origin of retroviral oncogenes”.