David G. Stokes

Nucleus pulposus cells express HIF‐1α under normoxic culture conditions: A metabolic adaptation to the intervertebral disc microenvironment

Nucleus pulposus (NP) cells of the intervertebral disc reside in an environment that has a limited vascular supply and generate energy through anaerobic glycolysis. The goal of the present study was to examine the expression and regulation of HIF‐1α, a transcription factor that regulates oxidative metabolism in nucleus pulposus cells. Nucleus pulposus cells were isolated from rat, human, and sheep disc and maintained at either 21% or 2% oxygen for various time periods. Cells were also treated with desferrioxamine (Dfx), a compound that mimics the effects of hypoxia (Hx). Expression and function of HIF‐1α were assessed by immunofluorescence microscopy, Western blot analysis, gel shift assays, and luciferase reporter assays. In normoxia (Nx), rat, sheep, and human nucleus pulposus cells consistently expressed the HIF‐1α subunit. Unlike other skeletal cells, when maintained under low oxygen tension, the nucleus pulposus cells exhibited a minimal induction in HIF‐1α protein levels. Electromobility shift assays confirmed the functional binding of normoxic HIF‐1α protein to its putative DNA binding motif. A dual luciferase reporter assay showed increased HIF‐1α transcriptional activity under hypoxia compared to normoxic level, although this induction was small when compared to HeLa and other cell types. These results indicate that normoxic stabilization of HIF‐1α is a metabolic adaptation of nucleus pulposus cells to a unique oxygen‐limited microenvironment. The study confirmed that HIF‐1α can be used as a phenotypic marker of nucleus pulposus cells. J. Cell. Biochem. 98: 152–159, 2006.

Regulation of type-II collagen gene expression during human chondrocyte de-differentiation and recovery of chondrocyte-specific phenotype in culture involves Sry-type high-mobility-group box (SOX) transcription factors.

During ex vivo growth as monolayer cultures, chondrocytes proliferate and undergo a process of de-differentiation. This process involves a change in morphology and a change from expression of chondrocyte-specific genes to that of genes that are normally expressed in fibroblasts. Transfer of the monolayer chondrocyte culture to three-dimensional culture systems induces the cells to re-acquire a chondrocyte-specific phenotype and produce a cartilaginous-like tissue in vitro. We investigated mechanisms involved in the control of the de-differentiation and re-differentiation process in vitro. De-differentiated chondrocytes re-acquired their chondrocyte-specific phenotype when cultured on poly-(2-hydroxyethyl methacrylate) (polyHEMA) as assayed by morphology, reverse transcriptase PCR of chondrocyte-specific mRNA, Western-blot analysis and chondrocyte-specific promoter activity. Essentially, full recovery of the chondrocyte-specific phenotype was observed when cells that had been cultured for 4 weeks on plastic were transferred to culture on polyHEMA. However, after subsequent passages on plastic, the phenotype recovery was incomplete or did not occur. The activity of a gene reporter construct containing the promoter and enhancer from the human type-II collagen gene (COL2A1) was modulated by the culture conditions, so that its transcriptional activity was repressed in monolayer cultures and rescued to some extent when the cells were switched to polyHEMA cultures. The binding of Sry-type high-mobility-group box (SOX) transcription factors to the enhancer region was modulated by the culture conditions, as were the mRNA levels for SOX9. A transfected human type-II collagen reporter construct was activated in de-differentiated cells by ectopic expression of SOX transcription factors. These results underscore the overt change in phenotype that occurs when chondrocytes are cultured as monolayers on tissue-culture plastic substrata.

Human clathrin heavy chain (CLTC): Partial molecular cloning, expression, and mapping of the gene to human chromosome 17q11-qter

The nucleotide sequence of a 916-bp human cDNA clone isolated from a human colon lambda gt11 cDNA library was determined. Sequence analysis showed this cDNA to have 88% homology to the nucleotide sequence of the heavy chain of rat clathrin. The deduced amino acid sequence was 98.7% identical to the rat sequence, a change of only four amino acids. The mRNA identified in both human and rat cells with the human clathrin clone revealed transcripts of approximately 6.5 kb, which is consistent with the predicted 180 kDa molecular weight of the clathrin heavy chain. Southern analysis of human/rodent somatic cell hybrids localized the human clathrin heavy chain gene (CLTC) to chromosome 17. Additional analyses using panels of human/rodent somatic cell hybrids with specific chromosomal translocations and deletions mapped the human clathrin heavy chain gene locus to 17q11-qter.

P5L mutation in Ank results in an increase in extracellular inorganic pyrophosphate during proliferation and nonmineralizing hypertrophy in stably transduced ATDC5 cells

Ank is a multipass transmembrane protein that regulates the cellular transport of inorganic pyrophosphate. In the progressive ankylosis (ank) mouse, a premature termination mutation at glutamic acid 440 results in a phenotype characterized by inappropriate deposition of basic calcium phosphate crystals in skeletal tissues. Mutations in the amino terminus of ANKH, the human homolog of Ank, result in familial calcium pyrophosphate dihydrate deposition disease. It has been hypothesized that these mutations result in a gain-of-function with respect to the elaboration of extracellular inorganic pyrophosphate. To explore this issue in a mineralization-competent system, we stably transduced ATDC5 cells with wild-type Ank as well as with familial chondrocalcinosis-causing Ank mutations. We evaluated the elaboration of inorganic pyrophosphate, the activity of pyrophosphate-modulating enzymes, and the mineralization in the transduced cells. Expression of transduced protein was confirmed by quantitative real-time PCR and by ELISA. Levels of inorganic pyrophosphate were measured, as were the activities of nucleotide pyrophosphatase phosphodiesterase and alkaline phosphatase. We also evaluated the expression of markers of chondrocyte maturation and the nature of the mineralization phase elaborated by transduced cells. The cell line expressing the proline to leucine mutation at position 5 (P5L) consistently displayed higher levels of extracellular inorganic pyrophosphate and higher phosphodiesterase activity than the other transduced lines. During hypertrophy, however, extracellular inorganic pyrophosphate levels were modulated by alkaline phosphatase activity in this cell system, resulting in the deposition of basic calcium phosphate crystals only in all transduced cell lines. Cells overexpressing wild-type Ank displayed a higher level of expression of type X collagen than cells transduced with mutant Ank. Other markers of hypertrophy and terminal differentiation, such as alkaline phosphatase, osteopontin, and runx2, were not significantly different in cells expressing wild-type or mutant Ank in comparison with cells transduced with an empty vector or with untransduced cells. These results suggest that the P5L Ank mutant is capable of demonstrating a gain-of-function with respect to extracellular inorganic pyrophosphate elaboration, but this effect is modified by high levels of expression of alkaline phosphatase in ATDC5 cells during hypertrophy and terminal differentiation, resulting in the deposition of basic calcium phosphate crystals.

Stable transfection of human fetal chondrocytes with a type II procollagen minigene : Expression of the mutant protein and alterations in the structure of the extracellular matrix in vitro

OBJECTIVE To perform stable transfections of human chondrocytes under conditions that allow the maintenance of the chondrocyte-specific phenotype, and to examine the effects of the stable transfection of a mutated type II procollagen gene (COL2A1) on the structure of the cartilaginous extracellular matrix produced in vitro. METHODS A type II procollagen minigene that lacks exons 16-27 was stably transfected into human fetal epiphyseal chondrocytes in vitro. Expression of the minigene was detected by reverse transcriptase-polymerase chain reaction, and the encoded protein was identified by Western blot with a human type II collagen-specific antibody. The cartilaginous extracellular matrix produced by the cultured transfected chondrocytes was characterized using histochemical staining, polarized light microscopy analysis, and transmission electron microscopy. RESULTS A shortened type II collagen encoded by the transfected minigene was biosynthesized and produced in the cultures of transfected cells. Histologic analyses demonstrated a more dense, negatively charged cartilaginous matrix in control cultures. In contrast, COL2A1 minigene-transfected cultures were more cellular, were populated with cells of irregular shape and less-chondrocytic appearance, contained abundant intracellular dense granules, and were surrounded by a less-dense matrix. Polarized light microscopy and transmission electron microscopy revealed a well-organized collagen fibrillar matrix in untransfected, control chondrocyte cultures, while the matrix in the transfected cultures was less birefringent and contained numerous truncated collagen fibrils. CONCLUSION The findings illustrate the feasibility of gene transfer into human fetal chondrocytes under conditions that allow the preservation of their specific phenotype, and also shed light on the function of type II collagen in the maintenance of the structural integrity of articular cartilage matrix.