Role of Cyr61/CCN1 in Mesenchymal Stem Cell Niche and Aging Bone

South Texas Veterans Health Care System, San Antonio TX

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Abstract

Aging-related skeletal degeneration is associated with changes in bone microarchitecture, loss of bone mineral density (BMD), increased susceptibility to fracture, and delayed bone healing. A key factor in these degenerative changes is the formation of osteoprogenitors (i.e. mesenchymal stem cells [MSCs]), which decreases with aging and are regulated by cues in the local bone marrow (BM) microenvironment (niche). To date, the specific changes that occur in the BM niche during aging are unknown. To address this knowledge gap, Dr. Xiao-Dong Chen's lab developed a culture system that reproduces the BM-MSC niche ex vivo, used in this project to compare the growth factor responsiveness of MSCs cultured on ECMs produced by BM stromal cells from “young” (≤25 y/o) and “old” (≥60 y/o) donors. MSCs on “old” ECM displayed less BMP-2 responsiveness compared to cells on “young” ECM. Recent preliminary data showed that Cyr61/CCN1, a matricellular protein involved in regulating osteogenesis, was deficient in “old” compared to “young” ECM and knock-down of Cyr61 in young ECM abrogated BMP-2 responsiveness, confirming the importance of this protein in osteogenesis. Further, treating old BM stromal cells with adenovirus containing the Cyr61 gene prior to ECM synthesis restored MSC responsiveness to BMP-2 and studies by Zhao et al (2018) using Cyr61 KO mice (i.e. Cyr61 KO [yKO]) driven by osterix (Osx) showed that KO mice had reduced BMD relative to wild type (WT). These findings lead to the hypothesis that aging-related bone degeneration is at least partially related to depletion of Cyr61 in bone ECM, which negatively impacts the BM niche and reduces MSC osteogenesis. In the proposed studies, Aim 1 will assess the impact of Cyr61 depletion on bone phenotype and BM matrix proteome in 3 mo. old (WT-y), 18 mo. old (WT-o) and 3 mo. old KO (yKO) mice. The bone matrix of WT-o and yKO mice is expected to contain less Cyr61, exhibit similar aging proteomes, and reduced BMD as compared to WT-y mice. Aim 2 will assess the ability of ECMs, produced by BM stromal cells from WT-y, WT-o and yKO mice, to support MSC growth factor responsiveness to BMP-2 and IGF-1 and osteoblast differentiation. These studies are expected to show that matrix bound Cyr61 determines the ability of an ECM to support MSC responsiveness to growth factors and osteoblast differentiation. Studies to probe the mechanism of aging-related changes in Cyr61 are expected to show that higher levels of active YAP [i.e. de- phosphorylated] in young MSCs promote the expression of Cyr61 and its increased incorporation into young BM-ECM. In contrast, old MSCs are expected to show lower levels of YAP and reduced Cyr61 in the ECM. Aim 3 will determine if exogenous rhCyr61 promotes lumbar fusion in WT-o and yKO mice, either alone or in combination with rhBMP-2. The results are expected to show that co-administration of rhCyr61 and rhBMP-2 will dramatically improve bone healing, better than either one alone, especially in WT-o and yKO mice. The results of Aim 3 have high translation potential as supplementing BMP-2 with Cyr61 will reduce the dose of BMP-2 necessary to achieve fusion and decrease its side-effects. Overall, the results of this project have the potential to significantly improve Veteran health care by providing an innovative approach for regenerating/healing bone under conditions where this is difficult to achieve (i.e. aging- related diseases and co-morbidities). Moreover, the proposal contains a well-developed plan for gaining new skills in bone and aging biology, proteomics, bioinformatics, and small animal surgery and targeted career development to become an independent VA research investigator.