John A. Feild

Cathepsin K mRNA Detection Is Restricted to Osteoclasts During Fetal Mouse Development

We recently identified a novel cysteine protease, cathepsin K, by random sequencing of an osteoclast cDNA library, and in situ hybridization studies in adult human tissues demonstrated high and specific expression in osteoclasts. To determine whether the expression of cathepsin K mRNA during mouse embryogenesis was more widespread, cryostat sections of early (day 11–13) and late (day 15–17) mouse fetuses were analyzed by in situ hybridization. Serial cross‐sections were collected through each fetus, and co‐reacted for tartrate‐resistant acid phosphatase (TRAP) and nonspecific esterase (NSE), selective markers for the osteoclast, and precursor cells derived from the macrophage/monocyte lineage, respectively. In the 11–13 day fetuses, cathepsin K mRNA was not expressed in any extraskeletal tissue; at this stage of embryogenesis, no osteoclasts are present. However, in the 15–17 day fetuses, a distinctive, developmental stage‐dependent pattern of cathepsin K expression was observed in osteoclasts and preosteoclasts at sites of cartilage and bone modeling. Cathepsin K positive osteoclasts differentiated within a peripheral zone of the osteogenic stacked cell layer of the cartilage rudiments (prior to ossification), migrated and/or resorbed the bone collar, and invaded the cartilage core. Furthermore, following the invasive penetration of vasculature into the degenerating cartilage core, the calcified cartilage was resorbed by cathepsin K positive mononuclear osteoclast precursors (NSE+ve, negligible TRAP); cells positive for both enzymes were identified indicative of osteoclast differentiation. The deposition of bone by osteoblasts onto the cartilage remnants is followed by mononucleated and multinucleated osteoclastic resorption; these osteoclasts demonstrated intense cathepsin K expression. Similar expression patterns were observed at sites of intramembranous ossification. No expression was observed in chondrocytes, osteoblasts, marrow, or in any other nonskeletal tissue at these time points. These data indicated that cathepsin K expression during embryogenesis occurred only following the onset of osteoclast differentiation.

Cloning and Characterization of a Novel Integrin β3Subunit

We have identified a novel integrin β3 subunit, termed β3C, from a human osteoclast cDNA library. The COOH-terminal sequence and 3′-untranslated region of the β3C subunit differs from the previously reported β3A (platelet) and β3B (placenta) sequences, while the regions coding for the transmembrane and extracellular domains are identical. The β3C cytoplasmic domain contains 37 amino acids, the last 17 of which are encoded by a novel exon located about 6 kilobase pairs downstream of exon 14 of the β3A gene. HEK 293 cells were stably co-transfected with αV and either β3C (HEKβ3C) or β3A(HEKβ3A). The viability of HEKβ3C cells was lower than that of HEKβ3A cells, and HEKβ3Ccells in culture grew as clusters rather than as a monolayer. The novel cytoplasmic domain did not affect receptor binding affinity; both αVβ3A and αVβ3Cisoforms exhibited high affinity binding to 125I-echistatin and cyclic and linear RGD peptides. However, in contrast to HEKβ3A, HEKβ3C cells failed to adhere to osteopontin, an αVβ3 matrix protein. The data provide further support for the key role of the cytoplasmic domain of the β3 integrin in cell adhesion and suggest a potential role for the β3C integrin subunit in modulating cell-matrix interactions.

Molecular and pharmacological characterization of the murine tachykinin NK3 receptor

Starting with a partial sequence from Genbank, polymerase chain reaction (PCR) was utilized to isolate the full-length cDNA for NK(3) receptor from mouse brain. The murine NK(3) receptor has a predicted sequence of 452 amino acids, sharing 96% and 86% identity to the rat and human NK(3) receptors, respectively. Binding affinities and functional potencies of tachykinin receptor agonists were similar in HEK (human embryonic kidney) 293 cells expressing murine NK(3) receptor and human NK(3) receptor, although substance P and neurokinin A were more potent stimulators of Ca(2+) mobilization in murine NK(3) receptor cells. NK(3) receptor-selective antagonists from two structural classes, had 10- to 100-fold lower binding affinities for murine NK(3) receptor compared to human NK(3) receptor, and about 5- to 10-fold reduced potency in the murine NK(3) receptor functional assay. The results demonstrate species differences in the potencies of tachykinin receptor antagonists in murine and human NK(3) receptors, and the lower potencies in the former should be taken into consideration when using murine disease models.

Biological and biophysical characterization of recombinant soluble human E-selectin purified at large scale by reversed-phase high-performance liquid chromatography

A first step in the development of a high-throughput screening assay for antagonists of human E-selectin is the purification and characterization of the selectin. In the present paper we describe a single-step, rapid, reversed-phase HPLC purification protocol for the recombinant, soluble form of human E-selectin (rshE-selectin) produced in Chinese hamster ovary cells. The procedure resulted in high protein yields with recoveries of greater than 98%. Characterization of the reversed-phase purified rshE-selectin showed this product to be analogous to rshE-selectin purified using conventional chromatographic techniques with respect to biological activity and molecular shape. However, the carbohydrate composition of reversed-phase purified rshE-selectin, which had been variable with conventionally purified material, was found to be constant across several isolations. The protocol described herein eliminated the high mannose component associated with previously purified rshE-selectin and provided a uniform carbohydrate composition for additional experimental studies, such as NMR. This fact, coupled with the high yield and simplicity of the present purification scheme are distinct advantages over those previously published. It is expected that other mammalian selectins, such as P-selectin and L-selectin, would also be amenable to reversed-phase HPLC purification.

Cloning and characterization of a rabbit ortholog of human Gα16 and mouse Gα15

A cDNA was cloned from a rabbit spleen cDNA library which encoded a G‐protein α subunit peptide of 374 amino acids, that at the peptide level exhibited 86% and 79% identity with human Gα16 and mouse Gα15, respectively. The rabbit Gα subunit cDNA was subcloned into a mammalian expression vector and transiently co‐transfected into HEK‐293 cells along with cDNAs encoding the human C3a, C5a, or nociceptin/orphanin FQ receptors. In all three cases the rabbit G α subunit behaved similarly to Gα15 or Gα16 and effectively coupled the transfected receptors to intracellular calcium mobilization pathways. By nucleotide sequence homology and functional activity the rabbit Gα subunit appears to be the ortholog of human Gα16 and mouse Gα15.