Modeled microgravity disrupts collagen I/integrin signaling during osteoblastic differentiation of human mesenchymal stem cells

Abstract

2Spaceflight leads to reduced bone mineral density in weight bearing bones that is primarily attributed to a reduction in bone formation. We have previously demonstrated severely reduced osteoblastogenesis of human mesenchymal stem cells (hMSC) following seven days culture in modeled microgravity. One potential mechanism for reduced osteoblastic differentiation is disruption of type I collagen-integrin interactions and reduced integrin signaling. lntegrins are heterodimeric transmembrane receptors that bind extracellular matrix proteins and produce signals essential for proper cellular function, survival, and differentiation. Therefore, we investigated the effects of modeled microgravity on integrin expression and function in hMSC.We demonstrate that seven days of culture in modeled microgravity leads to reduced expression of the extracellular matrix protein, type I collagen (Col I). Conversely, modeled microgravity consistently increases Col I-specific a2 and PI integrin protein expression. Despite this increase in integrin subunit expression, autophosphorylatior! of adhesion-dependent kinases, focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 ( P Y U ) , is significantly reduced. Activation of Akt is unaffected by the reduction in FAK activation. However, reduced downstream signaling via the Ras-MAPK pathway is evidenced by a reduction in Ras and ERK activation. Taken together, our findings indicate that modeled microgravity decreases integrin/MAPK signaling, which likely contributes to the observed reduction in osteoblastogenesis. 3Altered human bone homeostasis during spaceflight results in severe decreases in bone mineral density, the severity of which directly correlates with flight duration [Bikle and Halloran, 19991. It has been estimated that 1-2% site-specific bone loss occurs in the human skeleton each month during spaceflight [Tilton et al., 19801. Investigations of the potential mechanisms underlying this phenomenon will provide insight into the causes of disuse and age-related osteoporosis. Several independent studies of humans during spaceflight have detected decreased serum levels of bone formation markers, including alkaline phosphatase, osteocalcin, and the C-terminal peptide of pro-collagen type I [Caillot-Augusseau et al., 2000; Collet et al., 19971. Previous in vitro studies of osteosarcoma cells in microgravity have also identified a reduction in markers of osteoblast function, including type I collagen, alkaline phosphatase, and osteocalcin gene expression [Carmeliet et al., 1998; Landis et al., 20001. Due to payload constraints, flight frequency, and cost, spaceflight experiments are limited. Therefore, NASA has developed a commercially available rotary cell culture system (RCCS) to model microgravity in ground-based experiments. We have previously used and described this system in detail [Zayzafoon et al., 20041. The high aspect ratio vessels (HARVs), used in the RCCS, provide two essential components of optimized suspension culture: 1) Solid body rotation an...

Exposure to modeled microgravity induces metabolic idleness in malignant human MCF‐7 and normal murine VSMC cells

“…Since digital densitometry integrates bands produced on films by chemiluminescent signals, which are intrinsically semi-quantitative, the data should be regarded with some caution: nonetheless, they are not in contrast with our metabolic and cytofluorimetric findings (see below) and previous observations made by others [10,11].…”

Heterogeneous Differentiation of Human Mesenchymal Stem Cells in Response to Extended Culture in Extracellular Matrices

“…In addition, ECM proteins harbor integrin ligands that when bound promote adhesion and induce cellular signaling, including signals that drive cell fate processes. [5][6][7] The effect of cell-ECM interactions on cell fate processes has been thoroughly described for mature cells 8 but has only recently been examined for stem cells. 9 Exploration of these interactions is crucial for directing or avoiding differentiation.…”

Proteomics and genomics of microgravity

“…Human mesenchymal stem cells (hMSC) failed to display detectable levels for mRNA for major osteoblastic markers, including ALP, osteonectin, procollagen type I, and runx2 after 7 days in simulated microgravity conditions, despite osteogenic induction (121). Collagen type I, the most abundant protein in the extracellular matrix of bone, was also dramatically reduced, while collagen-binding ␣ 2 -and ␤ 1 -integrin subunit expression was increased following 7 days in modeled microgravity (72). However, integrin signaling through focal adhesion protein (FAK), Ras, and extracellular signal-regulated kinase (ERK) was significantly reduced.…”

“…However, integrin signaling through focal adhesion protein (FAK), Ras, and extracellular signal-regulated kinase (ERK) was significantly reduced. Due to the potential role for FAK in osteoblast differentiation through the MAPK pathway (106), which signals ERK, essential for osteoblast differentiation (61), leading to runx2 activation, the reduction in integrin signaling likely contributes to reduced osteoblastogenesis (72). Suppression of RhoA in hMSC cultured in a rotary cell culture system has also been suggested to play a role in reduced osteoblastogenesis and enhanced adipogenesis in hMSC (71).…”