HIF1α is a central regulator of collagen hydroxylation and secretion under hypoxia during bone development.

Collagen production is fundamental for the ontogeny and the phylogeny of all multicellular organisms. It depends on hydroxylation of proline residues, a reaction that uses molecular oxygen as a substrate. This dependency is expected to limit collagen production to oxygenated cells. However, during embryogenesis, cells in different tissues that develop under low oxygen levels must produce this essential protein. In this study, using the growth plate of developing bones as a model system, we identify the transcription factor hypoxia-inducible factor 1 α (HIF1α) as a central component in a mechanism that underlies collagen hydroxylation and secretion by hypoxic cells. We show that Hif1a loss of function in growth plate chondrocytes arrests the secretion of extracellular matrix proteins, including collagen type II. Reduced collagen hydroxylation and endoplasmic reticulum stress induction in Hif1a-depleted cells suggests that HIF1α regulates collagen secretion by mediating its hydroxylation and consequently its folding. We demonstrate in vivo the ability of Hif1α to drive the transcription of collagen prolyl 4-hydroxylase, which catalyzes collagen hydroxylation. We also show that, concurrently, HIF1α maintains cellular levels of oxygen, most likely by controlling the expression of pyruvate dehydrogenase kinase 1, an inhibitor of the tricarboxylic acid cycle. Through this two-armed mechanism, HIF1α acts as a central regulator of collagen production that allows chondrocytes to maintain their function as professional secretory cells in the hypoxic growth plate. As hypoxic conditions occur also during pathological conditions such as cancer, our findings may promote the understanding not only of embryogenesis, but also of pathological processes.

HIF1alpha regulation of Sox9 is necessary to maintain differentiation of hypoxic prechondrogenic cells during early skeletogenesis.

During early stages of limb development, the vasculature is subjected to extensive remodeling that leaves the prechondrogenic condensation avascular and, as we demonstrate hereafter, hypoxic. Numerous studies on a variety of cell types have reported that hypoxia has an inhibitory effect on cell differentiation. In order to investigate the mechanism that supports chondrocyte differentiation under hypoxic conditions, we inactivated the transcription factor hypoxia-inducible factor 1alpha (HIF1alpha) in mouse limb bud mesenchyme. Developmental analysis of Hif1alpha-depleted limbs revealed abnormal cartilage and joint formation in the autopod, suggesting that HIF1alpha is part of a mechanism that regulates the differentiation of hypoxic prechondrogenic cells. Dramatically reduced cartilage formation in Hif1alpha-depleted micromass culture cells under hypoxia provided further support for the regulatory role of HIF1alpha in chondrogenesis. Reduced expression of Sox9, a key regulator of chondrocyte differentiation, followed by reduction of Sox6, collagen type II and aggrecan in Hif1alpha-depleted limbs raised the possibility that HIF1alpha regulation of Sox9 is necessary under hypoxic conditions for differentiation of prechondrogenic cells to chondrocytes. To study this possibility, we targeted Hif1alpha expression in micromass cultures. Under hypoxic conditions, Sox9 expression was increased twofold relative to its expression in normoxic condition; this increment was lost in the Hif1alpha-depleted cells. Chromatin immunoprecipitation demonstrated direct binding of HIF1alpha to the Sox9 promoter, thus supporting direct regulation of HIF1alpha on Sox9 expression. This work establishes for the first time HIF1alpha as a key component in the genetic program that regulates chondrogenesis by regulating Sox9 expression in hypoxic prechondrogenic cells.

Differential regulation of endoplasmic reticulum structure through VAP-Nir protein interaction.

The endoplasmic reticulum (ER) exhibits a characteristic tubular structure that is dynamically rearranged in response to specific physiological demands. However, the mechanisms by which the ER maintains its characteristic structure are largely unknown. Here we show that the integral ER-membrane protein VAP-B causes a striking rearrangement of the ER through interaction with the Nir2 and Nir3 proteins. We provide evidence that Nir (Nir1, Nir2, and Nir3)-VAP-B interactions are mediated through the conserved FFAT (two phenylalanines (FF) in acidic tract) motif present in Nir proteins. However, each interaction affects the structural integrity of the ER differently. Whereas the Nir2-VAP-B interaction induces the formation of stacked ER membrane arrays, the Nir3-VAP-B interaction leads to a gross remodeling of the ER and the bundling of thick microtubules along the altered ER membranes. In contrast, the Nir1-VAP-B interaction has no apparent effect on ER structure. We also show that the Nir2-VAP-B interaction attenuates protein export from the ER. These results demonstrate new mechanisms for the regulation of ER structure, all of which are mediated through interaction with an identical integral ER-membrane protein.

Mitotic phosphorylation of the peripheral Golgi protein Nir2 by Cdk1 provides a docking mechanism for Plk1 and affects cytokinesis completion.

The rearrangement of the Golgi apparatus during mitosis is regulated by several protein kinases, including Cdk1 and Plk1. Several peripheral Golgi proteins that dissociate from the Golgi during mitosis are implicated in regulation of cytokinesis or chromosome segregation, thereby coordinating mitotic and cytokinetic events to Golgi rearrangement. Here we show that, at the onset of mitosis, Cdk1 phosphorylates the peripheral Golgi protein Nir2 at multiple sites; of these, S382 is the most prominent. Phosphorylation of Nir2 by Cdk1 facilitates its dissociation from the Golgi apparatus, and phospho-Nir2(pS382) is localized in the cleavage furrow and midbody during cytokinesis. Mitotic phosphorylation of Nir2 is required for docking of the phospho-Ser/Thr binding module, the Polo box domain of Plk1, and overexpression of a Nir2 mutant, which fails to interact with Plk1, affects the completion of cytokinesis. These results demonstrate a mechanism for coordinating mitotic and cytokinetic events with Golgi rearrangement during cell division.

Targeting of Nir2 to lipid droplets is regulated by a specific threonine residue within its PI-transfer domain.

Nir2, like its Drosophila homolog retinal degeneration B (RdgB), contains an N-terminal phosphatidylinositol-transfer protein (PI-TP)-like domain. Previous studies have suggested that RdgB plays an important role in the fly phototransduction cascade and that its PI-transfer domain is critical for this function. In this domain, a specific mutation, T59E, induces a dominant retinal degeneration phenotype. Here we show that a similar mutation, T59E in the human Nir2 protein, targets Nir2 to spherical cytosolic structures identified as lipid droplets by the lipophilic dye Nile red. A truncated Nir2T59E mutant consisting of only the PI-transfer domain was also targeted to lipid droplets, whereas neither the wild-type Nir2 nor the Nir2T59A mutant was associated with lipid droplets under regular growth conditions. However, oleic-acid treatment caused translocation of wild-type Nir2, but not translocation of the T59A mutant, to lipid droplets. This treatment also induced partial targeting of endogenous Nir2, which is mainly associated with the Golgi apparatus, to lipid droplets. Targeting of Nir2 to lipid droplets was attributed to its enhanced threonine phosphorylation. These results suggest that a specific threonine within the PI-transfer domain of Nir2 provides a regulatory site for targeting to lipid droplets. In conjunction with the role of PI-TPs in lipid transport, this targeting may affect intracellular lipid trafficking and distribution and may provide the molecular basis underlying the dominant effect of the RdgB-T59E mutant on retinal degeneration.