In this project, we have continued our efforts to establish how the skeleton and in particular the skeletal stem/progenitor population is maintained and differentiates within the confines of an actively remodeling extracellular matrix. To this end, we have extended our work to understand how the various progeny of the skeletal stem cells and osteoclastic cells, identified by distinct differentiation stage-specific gene expression rely on Mt-Mmp-dependent proteolysis for both physical matrix remodeling as well as modulation of cell-signaling molecules to maintain skeletal homeostasis. 1. Osteoclast-specific expression of Mt1-Mmp augments osteoclast function in degrading unmineralized peri-osseous surfaces - Despite the dramatic role exerted by Mt1-Mmp in osteoprogenitors, the function in mature osteoclasts has remained ill defined despite the abundance of Mt1-Mmp expressed by these cells. Specific ablation of Mt1-Mmp activity in this hematopoietic cell population (by way of Lys2-Cre) confers a modest osteoprotective role on the trabecular bone compartment consistent with a loss of collagen degradation at neutral pH in osteoclasts devoid of Mt1-Mmp activity. The manuscript has been submitted to J Biol Chem (Kaplan et al, Bone). 2. Identity of Mt1-Mmp-dependent perivascular progenitors cells of the bone marrow - To define more specifically the parent progenitor or skeletal stem cell within the progenitor pool by surface markers, we have obtained cells from the marrow stroma of mice tagged for SM22alpha expression using SM22alphaCre activated beta-galactosidase activity. Subsequent X-gal staining has allowed us to trace the tissue contribution of the cells with beta-galactosidase activity. Using FACS and in vivo transplantation, we have demonstrated that CD106 (VCAM-1) positive cells from bone marrow stroma give rise to all skeletal compartments of ectopic ossicles generated by donor cells; i.e., bone lining cells, osteocytes, marrow adipocytes, marrow stroma and pericytes (the most primitive cell). In CD106, we have thus identified a reliable cell surface marker for procurement of stem progenitor cells in murine marrow, which contain SSCs. Mt1-Mmp deficiency results in dramatic alteration in the bone marrow environment associated with disruption of the bone formation. Notably, the absence of Mt1-Mmp expression in mature bone-associated cells (osteocalcin positive) drives a remarkable differentiation of SSCs into bone marrow adipocytes at the expense of hematopoietic marrow. To determine to what extent SM22-positive cells through their protease activity regulate adipogenic differentiation, we performed whole mount adipocyte contrasting and Q-microCT marrow fat quantification on bones derived from mice with Mt1-Mmp ablated in SM22alpha positive cell and control mice. Unlike in mice with selective loss of Mt1-Mmp in osteocalcin positive cells, the loss of Mt1-Mmp in earlier progenitors disables enzyme activity in the entire bone and marrow compartment. However, this does not translate into a dramatic expansion of marrow adipocytes in the bone of mice with selective loss of the enzyme in SM22alpha positive cells. These cells normally differentiate into cells that go on to express osteocalcin during the course of differentiation into mature bone cells as demonstrated by our X-gal-visualized lineage tracing. The absence of marrow adipocyte expansion thus suggests that the ablation of Mt1-Mmp at the early stage where SM22alpha is expressed leads to a wholesale loss of multipotent stroma-associated cells competent to differentiate into adipocytes and that this capacity is tied to their expression of Mt1-Mmp. Mt1-Mmp dependent regulation of Notch signaling in adipogenesis - Whereas loss of Mt1-Mmp-mediated proteolysis in skeletal progenitor cells (SM22alpha+) has profound effects on skeletal morphogenesis via both bone formation and resorption, loss of Mt1-Mmp function specifically in more mature progeny expressing the late marker of the osteoblast/osteocyte lineage, osteocalcin (Ocn), is less overt and importantly, not associated with excessive bone erosion. Loss of Mt1-Mmp activity in Ocn+ in the Ocn-Cre mice is, however, associated with a profound post-natal reduction in bone content, spontaneous fractures and rapid expansion of bone marrow adipocytes. SSCs/pericytes self-renew, and support hematopoiesis, and bone and adipocyte formation. The shift of progenitor differentiation into fat at the expense of bone and hematopoietic tissue is associated with an excessive upregulation of the non-canonical Notch receptor ligand, Dlk1/Pref-1. Dlk1 is membrane-bound and is expressed widely in connective tissues and some lymphoid cells, and it controls maturation of pre-B cells. Dlk1 KO mice exhibit systemic adipocyte expansion including in bone marrow, as well as diminished bone formation (collaboration with Drs. Bauer Heigedhy, FDA, manuscript about to be submitted). We thus observe a partial phenocopy in skeletal cells with either extremely high or no Dlk1 protein at all. Despite the differences in Dlk1 levels, the Dlk1 KO and Ocn-Cre mice both lack the bioactive soluble form of Dlk1, recognized as the form that mediates Dlk1 signaling. Consistent with the latter, we have demonstrated that membrane-type Dlk1 is a substrate of both Mt1-Mmp and Mt3-Mmp and both proteases will cleave within a region of residues 281-305 of Dlk1 producing a fragment equivalent to the soluble active form. We have demonstrated by co-culture experiments that processing of Dlk1 is an autocrine and not a juxtacrine processing event whereby mature bone cells shed soluble Dlk1 from their own surface. This molecule in turn is required for attenuation of adipogenic differentiation of the SSC pool. This signaling event leading to adipogenic differentiation is Notch receptor-dependent. In the absence of Mt1-Mmp-mediated shedding of Dlk1, the Notch dependent genes, Hes1 and Hey1, as well as Dlk1 itself are substantially upregulated. Consistent with this observation, we have shown that soluble epitope tagged Dlk1 significantly reduces the ability of the canonical Notch ligand, Delta-like 4 (Dll4), to activate Notch-dependent Rbpj transcription and in turn, Hes1 and Hey1. The precise function of Dlk1 in Notch signaling is at present poorly understood and we have initiated experiments to identify the binding partner for Dlk1 in bone marrow progenitors using our epitope tagged Dlk1. Although the partial homology between the canonical Notch ligands Delta/Jagged and Dlk1 makes the latter a possible binding partner of the Notch receptors, it does not directly induce receptor processing and subsequent signaling. It is possible that the action of Dlk1 is a function of non-activating Notch receptor occupation or steric interference with canonical ligand binding. Our observations demonstrate that the mature bone cell plays an essential role in determination of the fate of SSCs by control of adipogenic differentiation. As such, the mature bone cell is essential in maintenance of bone homeostasis through modulation of progenitor commitment.
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