Nucleus pulposus primary cilia alter their length in response to changes in extracellular osmolarity but do not control TonEBP-mediated osmoregulation.

The nucleus pulposus (NP) cells adapt to their physiologically hyperosmotic microenvironment through Tonicity-responsive enhancer binding protein (TonEBP/nuclear factor of activated T-cell5 [NFAT5])-mediated osmoregulation. Primary cilia in different organs serve diverse roles including osmosensing, but its contribution to NP cell osmoadaptive response is unknown. A high percentage of cultured primary NP cells possessed primary cilia that changed length in response to osmotic stimuli. Stable silencing of Intraflagellar Transport 88 (Ift88) or Kinesin Family Member 3 A (Kif3a) to inhibit the formation of primary cilia did not affect hyperosmotic upregulation of TonEBP. While ShKif3a blocked hyperosmotic increase of TonEBP-Transactivation Domain (TAD) activity, overall the knockdown of either gene did not alter the hyperosmotic status of proximal promoter activities and transcription of key TonEBP targets. On the other hand, a small decrease in TonEBP level under hypoosmotic condition was attenuated by Ift88 or Kif3a knockdown. Noteworthy, none of the TonEBP target genes were responsive to hypoosmotic stimulus in control and Ift88 or Kif3a knockdown cells, suggesting the primary role of TonEBP in the hyperosmotic adaptation of NP cells. Similarly, in Kif3a null mouse embryonic fibroblasts (MEFs), the overall TonEBP-dependent hyperosmotic responses were preserved. Unlike NP cells, TonEBP targets were responsive to hypoosmolarity in wild-type MEFs, and these responses remained intact in Kif3a null MEFs. Together, these results suggest that primary cilia are dispensable for TonEBP-dependent osmoadaptive response

Abcc6 Null Mice-a Model for Mineralization Disorder PXE Shows Vertebral Osteopenia Without Enhanced Intervertebral Disc Calcification With Aging

Chronic low back pain is a highly prevalent health condition intricately linked to intervertebral disc degeneration. One of the prominent features of disc degeneration that is commonly observed with aging is dystrophic calcification. ATP-binding cassette sub-family C member 6 (ABCC6), a presumed ATP efflux transporter, is a key regulator of systemic levels of the mineralization inhibitor pyrophosphate (PPi). Mutations in ABCC6 result in pseudoxanthoma elasticum (PXE), a progressive human metabolic disorder characterized by mineralization of the skin and elastic tissues. The implications of ABCC6 loss-of-function on pathological mineralization of structures in the spine, however, are unknown. Using the Abcc6 -/- mouse model of PXE, we investigated age-dependent changes in the vertebral bone and intervertebral disc. Abcc6 -/- mice exhibited diminished trabecular bone quality parameters at 7 months, which remained significantly lower than the wild-type mice at 18 months of age. Abcc6 -/- vertebrae showed increased TRAP staining along with decreased TNAP staining, suggesting an enhanced bone resorption as well as decreased bone formation. Surprisingly, however, loss of ABCC6 resulted only in a mild, aging disc phenotype without evidence of dystrophic mineralization. Finally, we tested the utility of oral K3Citrate to treat the vertebral phenotype since it is shown to regulate hydroxyapatite mechanical behavior. The treatment resulted in inhibition of the osteoclastic response and an early improvement in mechanical properties of the bone underscoring the promise of potassium citrate as a therapeutic agent. Our data suggest that although ectopic mineralization is tightly regulated in the disc, loss of ABCC6 compromises vertebral bone quality and dysregulates osteoblast-osteoclast coupling

GLUT1 Is Redundant in Hypoxic and Glycolytic Nucleus Pulposus Cells of the Intervertebral Disc

Glycolysis is central to homeostasis of nucleus pulposus (NP) cells in the avascular intervertebral disc. Since the glucose transporter, GLUT1, is a highly enriched phenotypic marker of NP cells, we hypothesized that it is vital for the development and postnatal maintenance of the disc. Surprisingly, primary NP cells treated with 2 well-characterized GLUT1 inhibitors maintained normal rates of glycolysis and ATP production, indicating intrinsic compensatory mechanisms. We showed in vitro that NP cells mitigated GLUT1 loss by rewiring glucose import through GLUT3. Of note, we demonstrated that substrates, such as glutamine and palmitate, did not compensate for glucose restriction resulting from dual inhibition of GLUT1/3, and inhibition compromised long-term cell viability. To investigate the redundancy of GLUT1 function in NP, we generated 2 NP-specific knockout mice: Krt19CreERT Glut1fl/fl and Foxa2Cre Glut1fl/fl. There were no apparent defects in postnatal disc health or development and maturation in mutant mice. Microarray analysis verified that GLUT1 loss did not cause transcriptomic alterations in the NP, supporting that cells are refractory to GLUT1 loss. These observations provide the first evidence to our knowledge of functional redundancy in GLUT transporters in the physiologically hypoxic intervertebral disc and underscore the importance of glucose as the indispensable substrate for NP cells

Dual Inhibition of Activin/Nodal/TGF-β and BMP Signaling Pathways by SB431542 and Dorsomorphin Induces Neuronal Differentiation of Human Adipose Derived Stem Cells

Damage to the nervous system can cause devastating diseases or musculoskeletal dysfunctions and transplantation of progenitor stem cells can be an excellent treatment option in this regard. Preclinical studies demonstrate that untreated stem cells, unlike stem cells activated to differentiate into neuronal lineage, do not survive in the neuronal tissues. Conventional methods of inducing neuronal differentiation of stem cells are complex and expensive. We therefore sought to determine if a simple, one-step, and cost effective method, previously reported to induce neuronal differentiation of embryonic stem cells and induced-pluripotent stem cells, can be applied to adult stem cells. Indeed, dual inhibition of activin/nodal/TGF-β and BMP pathways using SB431542 and dorsomorphin, respectively, induced neuronal differentiation of human adipose derived stem cells (hADSCs) as evidenced by formation of neurite extensions, protein expression of neuron-specific gamma enolase, and mRNA expression of neuron-specific transcription factors Sox1 and Pax6 and matured neuronal marker NF200. This process correlated with enhanced phosphorylation of p38, Erk1/2, PI3K, and Akt1/3. Additionally, in vitro subcutaneous implants of SB431542 and dorsomorphin treated hADSCs displayed significantly higher expression of active-axonal-growth-specific marker GAP43. Our data offers novel insights into cell-based therapies for the nervous system repair

BMP-non-responsive Sca1+ CD73+ CD44+ mouse bone marrow derived osteoprogenitor cells respond to combination of VEGF and BMP-6 to display enhanced osteoblastic differentiation and ectopic bone formation.

Clinical trials on fracture repair have challenged the effectiveness of bone morphogenetic proteins (BMPs) but suggest that delivery of mesenchymal stem cells (MSCs) might be beneficial. It has also been reported that BMPs could not increase mineralization in several MSCs populations, which adds ambiguity to the use of BMPs. However, an exogenous supply of MSCs combined with vascular endothelial growth factor (VEGF) and BMPs is reported to synergistically enhance fracture repair in animal models. To elucidate the mechanism of this synergy, we investigated the osteoblastic differentiation of cloned mouse bone marrow derived MSCs (D1 cells) in vitro in response to human recombinant proteins of VEGF, BMPs (-2, -4, -6, -9) and the combination of VEGF with BMP-6 (most potent BMP). We further investigated ectopic bone formation induced by MSCs pre-conditioned with VEGF, BMP-6 or both. No significant increase in mineralization, phosphorylation of Smads 1/5/8 and expression of the ALP, COL1A1 and osterix genes was observed upon addition of VEGF or BMPs alone to the cells in culture. The lack of CD105, Alk1 and Alk6 expression in D1 cells correlated with poor response to BMPs indicating that a greater care in the selection of MSCs is necessary. Interestingly, the combination of VEGF and BMP-6 significantly increased the expression of ALP, COL1A1 and osterix genes and D1 cells pre-conditioned with VEGF and BMP-6 induced greater bone formation in vivo than the non-conditioned control cells or the cells pre-conditioned with either VEGF or BMP-6 alone. This enhanced bone formation by MSCs correlated with higher CADM1 expression and OPG/RANKL ratio in the implants. Thus, combined action of VEGF and BMP on MSCs enhances osteoblastic differentiation of MSCs and increases their bone forming ability, which cannot be achieved through use of BMPs alone. This strategy can be effectively used for bone repair

The mitophagy receptor BNIP3 is critical for the regulation of metabolic homeostasis and mitochondrial function in the nucleus pulposus cells of the intervertebral disc

The contribution of mitochondria to the metabolic function of hypoxic NP cells has been overlooked. We have shown that NP cells contain networked mitochondria and that mitochondrial translocation of BNIP3 mediates hypoxia-induced mitophagy. However, whether BNIP3 also plays a role in governing mitochondrial function and metabolism in hypoxic NP cells is not known. BNIP3 knockdown altered mitochondrial morphology, and number, and increased mitophagy. Interestingly, BNIP3 deficiency in NP cells reduced glycolytic capacity reflected by lower production of lactate/H and lower ATP production rate. Widely targeted metabolic profiling and flux analysis using 1-2-C-glucose showed that the BNIP3 loss resulted in redirection of glycolytic flux into pentose phosphate and hexosamine biosynthesis as well as pyruvate resulting in increased TCA flux. An overall reduction in one-carbon metabolism was noted suggesting reduced biosynthesis. UC-glutamine flux analysis showed preservation of glutamine utilization to maintain TCA intermediates. The transcriptomic analysis of the BNIP3-deficient cells showed dysregulation of cellular functions including membrane and cytoskeletal integrity, ECM-growth factor signaling, and protein quality control with an overall increase in themes related to angiogenesis and innate immune response. Importantly, we observed strong thematic similarities with the transcriptome of a subset of human degenerative samples. Last, we noted increased autophagic flux, decreased disc height index and aberrant COL10A1/collagen X expression, signs of early disc degeneration in young adult knockout mice. These results suggested that in addition to mitophagy regulation, BNIP3 plays a role in maintaining mitochondrial function and metabolism, and dysregulation of mitochondrial homeostasis could promote disc degeneration. ECAR extracellular acidification rate; HIF hypoxia inducible factor; MFA metabolic flux analysis; NP nucleus pulposus; OCR oxygen consumption rate; Sh short-hairpin

Preconditioning of BMP-non-responsive D1 cells with VEGF and BMP-6 enhances their ability to induce ectopic bone formation.

<p>The bone density of harvested implants was measured by radiography and Image J software (A-C). Representative images are shown in the figure. H and E staining revealed typical histology of bone (E). Real time PCR showed modulation of RANKL/OPG ratio (D). * denotes p<0.05 compared with OM group at weeks 2.</p

Expression of ALP and COL1 genes and genes for transcription factors osterix and Dlx5 in the implants of D1 cells.

<p>mRNA expression was quantified using gene specific primers and real time PCR. cDNA prepared from RNA isolated from the harvested implants was used as the template. * denotes p<0.05 compared with OM group of the respective time point, unless indicated otherwise by the horizontal lines.</p

Combination of VEGF and BMP-6 enhances expression of osteogenic genes.

<p>Expression of ALP (A), COL1A1 (B), runx2 (C) and osterix (D) genes were determined by real time PCR at day 7. * denotes p<0.05.</p