Moreover, accumulating evidence in humans and in animal models now strongly suggest that senescence, and the associated alterations in cell signaling and increase in oxidative stress, constitute the physiological and molecular consequences of hormone deficiency and osteoporosis. Accelerated senescence syndromes caused by accumulation of unprocessed farnesylated nuclear lamina protein lamin-A (key in nuclear architecture), due to alterations in the lamin A gene (LMNA) mRNA splicing, show features of age-related bone loss. These include lower bone density and trabecular thickness, lower osteoblast and osteocytes numbers, impaired osteoblastic gene expression, differentiation and function, and higher levels of marrow adipogenesis. The LMNA mutation C1824T activates a cryptic splice site in exon 11 leading to the production of the unprocessed farnesylated LMNA form termed progerin. Progerin accumulation is characterized by increased levels of reactive oxygen species (ROS) leading to oxidative stress and DNA damage. ROS appears to contribute to decreased osteoblastogenesis and bone mass as well as quality. Progerin is elevated in skin fibroblasts of healthy older individuals due to sporadic use of the cryptic splice site in exon 11. Similarly, progerin accumulation in mesenchymal progenitor cells may signify a novel mechanism that contributes to osteoporosis. We propose that progerin accumulation in mesenchymal progenitor cells may be a novel mechanism that contributes to osteoporosis. Bone-forming osteoblasts originate from mesenchymal stromal cells (MSCs) in the bone marrow (BM). We have previously demonstrated an age-related decline in human MSCs (hMSCs) which correlates with decreased osteoblast numbers and age-related bone loss. Whether this age-related decline in hMSC is due to reduced self-renewal is not clear. Our data have shown that self-renewal of a developmentally immature population of hMSCs termed Marrow-Isolated Adult Multilineage Inducible (MIAMI) cells involves 2-catenin nuclear translocation and activation of the canonical Wnt signaling. To begin to address whether increased progerin has a role in reducing the osteogenic capacity of hMSCs, we generated MIAMI cells stably overexpressing mutant GFP-C1824T/LMNA (GFP-Progerin). The results showed that the accumulation of progerin decreased expression of self-renewal genes, increased expression of senescence- associated genes, increased DNA damage, and osteoblastic mRNA levels and biomineralization. New experimental evidence shows sporadic use of the LMNA cryptic splice site in exon 11 in MSCs from older individuals. These results strongly suggest that progerin alters hMSC behavior. Our hypothesis is that accumulation of progerin (unprocessed farnesylated lamin A) in hMSCs leads to a decreased osteogenic potential. We will employ readily available hMSCs and hMSC-GFP-Progerin cells to test our hypothesis by pursuing the following specific aims:
Aim 1 : Determine the role of progerin in the differentiation potential of hMSCs in vitro. We expect that increased progerin expression in hMSCs will decrease their osteoblastic capacity in vitro;and alter the binding of osterix to its target sites in the genome. We also expect that farnesyl-transferase inhibitors will attenuate these effects.
Aim 2 : Determine the role of progerin in nuclear shuttling and heterochromatinization of hMSCs in vitro. We expect that increased progerin expression in hMSCs will alter heterochromatin formation protein and translocation signaling molecules (i.e., transcription factors) to the nucleus, and the expression of antioxidant genes leading to increased ROS levels, oxidative stress and senescence. This will diminish the number of cells with bone forming potential. We expect that farnesyl-transferase inhibitors will attenuate these effects.
Aim 3 : Determine the role of progerin in the bone forming capacity of hMSCs in vivo. We expect that increased progerin expression in hMSCs will decrease their bone forming capacity in vivo.
The VA patient population is characterized by a high number of patients suffering from age-related degenerative diseases, not just in bone, but also in other tissues. The results obtained from the proposed work will be useful for increasing our understanding of the molecular and cellular events contributing to age-related osteoporosis and the repair of bone due to fractures, injuries, or other lesions. Successful translational research by developing strategies to prevent or decrease the senescence of mesenchymal progenitors for bone, and other tissues, could provide the basis to delay the development of age-related osteoporosis and address the applicability of cellular therapies and bioengineering for bone as well as other tissues resulting from age-related degenerative diseases. The proposed studies are designed to characterize the role of alterations in the nuclear membrane lamina in stem cell senescence and bone forming potential, and how to prevent them;and thus are vital to the VA patient care mission.
|Grau-Monge, Cristina; Delcroix, Gaëtan J-R; Bonnin-Marquez, Andrea et al. (2017) Marrow-isolated adult multilineage inducible cells embedded within a biologically-inspired construct promote recovery in a mouse model of peripheral vascular disease. Biomed Mater 12:015024|