The hypoxia signature across skeletal progenitor cells

Skeletal tissue serves as a dynamic hub of cellular activity orchestrated by a group of remarkable cells known as skeletal stem and progenitor cells (SSPCs). These versatile cells exhibit extraordinary abilities for self-renewal, proliferation, and differentiation into osteoblasts and chondrocytes, playing a pivotal role in bone development, maintenance, and repair.¹ The activity of SSPCs is intricately linked to the surrounding microenvironment, including oxygen availability, which can vary significantly within bone niches, often dropping to levels below 1%.²

In a study featured in this issue,³ Loopmans and colleagues have addressed an intriguing and timely question: whether, in the face of different environmental exposures to oxygen and nutrients, SSPCs share a uniform metabolic profile and respond consistently to the challenges posed by hypoxia. Their paper provides interesting results that pave the way for further exploration, offering a timely and critical step toward a comprehensive understanding of metabolism in skeletal cells.

Oxygen availability profoundly impacts cellular energy metabolism, shaping the core processes by which cells produce and utilize energy to maintain physiological functions and adapt to varying environmental conditions. Cells primarily employ 2 methods for energy production: glycolysis coupled with lactic acid fermentation in the cytosol and the tricarboxylic acid (TCA) cycle/oxidative phosphorylation (OxPhos) in mitochondria. Glycolysis can occur in the absence of oxygen, yielding a net production of 2 adenosine triphosphate (ATP) per cycle. In contrast, OxPhos relies on oxygen as the final electron acceptor in the electron transport chain, yielding a larger amount of ATP, typically ranging from 36 to 38 ATP per cycle. Both lactic acid fermentation and OxPhos regenerate NAD+ from NADH, a critical event driving both glycolysis and the TCA cycle.

A growing body of in vitro and in vivo data suggests that the choice between these energy sources during various phases of cellular differentiation may influence skeletal cell fate.⁴ Cell energy metabolism is no longer viewed as a mere consequence of cellular state. Instead, it has been proposed it actively regulates tissue differentiation and function with mechanisms that go beyond ATP production and include fine-tuning of ROS levels and the synthesis of intermediate metabolites controlling the epigenetic landscape.⁵

Fetal growth plate chondrocytes reside in a unique environment that is avascular and hypoxic.⁶ The adult bone marrow also displays a gradient of oxygenation despite its high degree of vascularization.⁷ The high cellularity of bone marrow, increased oxygen consumption by leukocytes, and sluggish blood flow in sinusoids may contribute to this oxygenation gradient. Comparable to the bone marrow, the periosteum shows significant vascularity, although precise information about periosteum oxygenation levels remains incomplete.

In their in vitro study, Loopmans and colleagues examined the response of various types of SSPCs, including neonatal SSPCs (nSSPCs), periosteal SSPCs (pSSPCs), and metaphyseal/endosteal SSPCs (meSSPCs), to hypoxic conditions and how these responses impact their cellular metabolism. Growth plate chondrocytes served as a prime example of cells adapted to a hypoxic microenvironment.

The study revealed that various cell types responded to hypoxia with similar metabolic adaptations, showcasing the impressive resilience of SSPCs. Hypoxia led to a significant increase in proliferation across all cell types. Furthermore, as reported for other cell types, hypoxia increased glycolysis and lactate fermentation, particularly in nSSPCs and chondrocytes. Additionally, hypoxia induced changes in the TCA cycle, with a reduction in glucose-derived citrate levels and a shift of pyruvate toward malate. This adaptation likely supports anaplerosis and plays a critical role in replenishing NAD+, essential for cell proliferation in low-oxygen environments. Hypoxia also induced the reductive carboxylation of glutamine in the cytosol, concurrently reducing oxidative decarboxylation in the mitochondria, further enhancing NAD+ availability. Amino acid metabolism underwent alterations, affecting components such as aspartate, serine, and glycine, which are fundamental in various cellular reactions. Finally, the activation of the pentose phosphate pathway under hypoxic conditions increased the production of glucose-derived nucleotides, including adenosine monophosphate and uridine monophosphate, potentially facilitating cell proliferation in low-oxygen conditions. Of note, though hypoxic adaptations generally exhibited a consistent pattern across the various skeletal cell types, yet meSSPCs manifested a distinctive metabolic signature marked by increased glucose-to-pyruvate conversion and pyruvate excretion. Conversely, hypoxic nSSPCs, pSSPCs, and chondrocytes used more glucose for alanine synthesis than hypoxic meSSPCs.

In summary, this research underscores the exceptional ability of skeletal SSPCs to reconfigure their metabolism in response to hypoxia. Further research is needed to fully appreciate and harness the biological relevance of those adaptations, particularly those that are cell specific.

A few challenging issues raised by the study are worthwhile discussing. Although in vitro conditions are indispensable for studying these cells, acknowledging that they are difficult to access and study in vivo, it need to be recognized that in vitro conditions may not fully replicate the in vivo situation. The latter is influenced by a complex interplay of microenvironmental factors that shape the behavior of these cells. Along those lines, as acknowledged by the authors, differently from the in vitro setting, hypoxia has been shown to result in a decrease in cell proliferation across various cell types including chondrocytes in vivo.^8,9 Furthermore, the observed metabolic changes in hypoxia, though strikingly similar across the different cell types, may differentially affect their biology depending on their unique genetic backgrounds, which inevitably vary from cell to cell.

Lastly, hypoxia activates the hypoxia signaling pathway with hypoxia-inducible factor 1 alpha (Hif1) being a critical factor in cellular adaptation to hypoxia. Hif1 is crucial for survival of growth plate chondrocytes by suppressing mitochondrial oxygen consumption and thus preventing naturally hypoxic chondrocytes from becoming virtually anoxic and dying.¹⁰ The finding raises the intriguing possibility that oxygen has vital functions in eukaryotic cells that go well beyond ATP production. A recent discovery has provided further support to this hypothesis by showing that chondrocytes store oxygen through Hif1-independent production and accumulation of hemoglobin, a molecule traditionally associated with red blood cells and oxygen transport.¹¹ This finding, in conjunction with prior knowledge, underscores, once again, the need for hypoxic chondrocytes of protecting themselves from lethal anoxia. Along those lines, in the future, it will be interesting to explore whether SSPCs metabolically adapt to hypoxia through Hif1-dependent or -independent mechanisms or both.

In conclusion, the study by Loopmans and colleagues unveils remarkable metabolic adaptations across different types of skeletal progenitors in response to hypoxia, including heightened proliferation, increased glycolysis and lactate fermentation, alterations in the TCA cycle, and shifts in amino acid metabolism. These findings not only advance our understanding of metabolic processes in skeletal cells but also provide a robust foundation for further investigations in this field.

Author contributions

Ernestina Schipani (Conceptualization, Data curation, Formal analysis) and Elena Sabini (Conceptualization, Data curation, Formal analysis).

Funding

This work was supported by the National Institutes of Health (NIH) under grant number R01 AR074079. The grant was awarded to Ernestina Schipani.

Conflicts of interest

The authors declare that there are no conflicts of interest regarding the publication of this article.