A key goal of molecular/cell biology/biotechnology is to identify essential genes in virtually every physiological process to uncover basic mechanisms of cell function and to establish potential targets of drug therapy combating human disease. This article describes a semester-long, project-oriented molecular/cellular/biotechnology laboratory providing students, within a framework of bone cell biology, with a modern approach to gene discovery. Students are introduced to the topics of bone cells, bone synthesis, bone resorption, and osteoporosis. They then review the theory of microchip gene arrays, and study microchip array data generated during the differentiation of bone-resorbing osteoclasts in vitro. The class selects genes whose expression increases during osteoclastogenesis, and researches them in small groups using web-based bioinformatics tools. Students then go to a biotechnology company website to find and order small inhibitory RNAs (siRNAs) designed to "knockdown'' expression of the gene of interest. Students then learn to transfect these siRNAs into osteoclasts, stimulate the cells to differentiate, assay osteoclast differentiation in vitro, and measure specific gene expression using real-time PCR and immunoblotting. Specific siRNA knockdown resulting in a decrease in osteoclastogenesis is indicative of a gene's physiological relevance. The results are analyzed statistically and presented to the class in groups. In the past 2 years, students identified several genes essential for optimal osteoclast differentiation, including Myo1d. The students hypothesize that the myo1d protein functions in osteoclasts to deliver important proteins to the cell surface via vesicular transport along microfilaments. Student response to the new course was overwhelmingly positive.

The septoclast is a specialized, cathepsin B-rich, perivascular cell type that accompanies invading capillaries on the metaphyseal side of the growth plate during endochondral bone growth. The putative role of septoclasts is to break down the terminal transverse septum of growth plate cartilage and permit capillaries to bud into the lower hypertrophic zone. This process fails in osteoclast-deficient, osteopetrotic animal models, resulting in a progressive growth plate dysplasia. The toothless rat is severely osteopetrotic because of a frameshift mutation in the colony-stimulating factor-1 (CSF-1) gene (Csf1(tl)). Whereas CSF-1 injections quickly restore endosteal osteoclast populations, they do not improve the chondrodysplasia. We therefore investigated septoclast populations in Csf1(tl)/Csf1(tl) rats and wild-type littermates, with and without CSF-1 treatment, at 2 weeks, before the dysplasia is pronounced, and at 4 weeks, by which time it is severe. Tibial sections were immunolabeled for cathepsin B and septoclasts were counted. Csf1(tl)/Csf1(tl) mutants had significant reductions in septoclasts at both times, although they were more pronounced at 4 weeks. CSF-1 injections increased counts in wild-type and mutant animals at both times, restoring mutants to normal levels at 2 weeks. In all of the mutants, septoclasts seemed misoriented and had abnormal ultrastructure. We conclude that CSF-1 promotes angiogenesis at the chondroosseous junction, but that, in Csf1(tl)/Csf1(tl) rats, septoclasts are unable to direct their degradative activity appropriately, implying a capillary guidance role for locally supplied CSF-1.

Bone morphogenetic proteins (BMPs) play pivotal roles in bone and cartilage growth and repair. Through phenotypes of short ‐ ear ( se ) mice, which have BMP‐5 mutations, a role for BMP‐5 in some specific aspects of skeletogenesis and cartilage growth is known. This report examines BMP‐5 expression in the growth plate and in differentiating cultures of primary chondrocytes, and the effects of addition of BMP‐5 or its inhibition by anti‐BMP‐5 antibody in chondrocyte cultures. By laser capture microdissection and immunohistochemistry, we found that BMP‐5 is expressed in proliferating zone (PZ) chondrocytes and that the expression increases sharply with hypertrophic differentiation. A similar pattern was observed in differentiating cultures of primary chondrocytes, with BMP‐5 expression increasing as cells differentiated, in contrast to other BMPs. BMP‐5 added to cultures increased cell proliferation early in the culture period and also stimulated cartilage matrix synthesis. Also, BMP‐5 addition to the cultures activated phosphorylation of Smad 1/5/8 and p38 MAP kinase and caused increased nuclear accumulation of phospho‐Smads. Anti‐BMP‐5 antibody inhibited the endogenous BMP‐5, reducing cell proliferation and phospho‐Smad nuclear accumulation. Together, the results demonstrate that BMP‐5 is normally an important regulator of chondrocyte proliferation and differentiation. Whether other BMPs may compensate in BMP‐5 loss‐of‐function mutations is discussed. J. Cell. Physiol. 214:56–64, 2008. © 2007 Wiley‐Liss, Inc.

Microarray and real‐time RT‐PCR were used to examine expression changes in primary bone marrow cells and RAW 264.7 cells in response to RANKL. In silico sequence analysis was performed on a novel gene which we designate OC‐STAMP. Specific siRNA and antibodies were used to inhibit OC‐STAMP RNA and protein, respectively, and tartrate‐resistant acid phosphatase (TRAP)+ multinucleated osteoclasts were counted. Antibodies were used to probe bone tissues and western blots of RAW cell extracts +/− RANKL. cDNA overexpression constructs were transfected into RAW cells and the effect on RANKL‐induced differentiation was studied. OC‐STAMP was very strongly up‐regulated during osteoclast differentiation. Northern blots and sequence analysis revealed two transcripts of 2 and 3.7 kb differing only in 3′UTR length, consistent with predictions from genome sequence. The mRNA encodes a 498 amino acid, multipass transmembrane protein that is highly conserved in mammals. It has little overall homology to other proteins. The carboxy‐terminal 193 amino acids, however, are significantly similar to the DC‐STAMP family consensus sequence. DC‐STAMP is a transmembrane protein required for osteoclast precursor fusion. Knockdown of OC‐STAMP mRNA by siRNA and protein inhibition by antibodies significantly suppressed the formation of TRAP+, multinucleated cells in differentiating osteoclast cultures, with many TRAP+ mononuclear cells present. Conversely, overexpression of OC‐STAMP increased osteoclastic differentiation of RAW 264.7 cells. We conclude that OC‐STAMP is a previously unknown, RANKL‐induced, multipass transmembrane protein that promotes the formation of multinucleated osteoclasts. J. Cell. Physiol. 215: 497–505, 2008. © 2007 Wiley‐Liss, Inc.

Osteoclasts differentiate from hematopoietic mononuclear precursor cells under the control of both colony stimulating factor-1 (CSF-1, or M-CSF) and receptor activator of NF-κB ligand (RANKL, or TRANCE, TNFSF11) to carry out bone resorption. Using high density gene microarrays, we followed gene expression changes in long bone RNA when CSF-1 injections were used to restore osteoclast populations in the CSF-1-null toothless (csf1tl/csf1tl) osteopetrotic rat. We found that ovarian cancer G-protein-coupled receptor 1 (OGR1, or GPR68) was strongly up-regulated, rising >6-fold in vivo after 2 days of CSF-1 treatments. OGR1 is a dual membrane receptor for both protons (extracellular pH) and lysolipids. Strong induction of OGR1 mRNA was also observed by microarray, real-time RT-PCR, and immunoblotting when mouse bone marrow mononuclear cells and RAW 264.7 pre-osteoclast-like cells were treated with RANKL to induce osteoclast differentiation. Anti-OGR1 immunofluorescence showed intense labeling of RANKL-treated RAW cells. The time course of OGR1 mRNA expression suggests that OGR1 induction is early but not immediate, peaking 2 days after inducing osteoclast differentiation both in vivo and in vitro. Specific inhibition of OGR1 by anti-OGR1 antibody and by small inhibitory RNA inhibited RANKL-induced differentiation of both mouse bone marrow mononuclear cells and RAW cells in vitro, as evidenced by a decrease in tartrate-resistant acid phosphatase-positive osteoclasts. Taken together, these data indicate that OGR1 is expressed early during osteoclastogenesis both in vivo and in vitro and plays a role in osteoclast differentiation.

Escherichia coli beta-galactosidase (beta-gal), encoded by the lacZ gene, has become an essential tool in studies of gene expression and function in higher eukaryotes. lac-Z is widely used as a marker gene to detect expression of transgenes or Cre recombinase driven by tissue-specific promoters. The timing and location of promoter activity is easily visualized in whole embryos or specific tissues using the cleavable, chromogenic substrate, 5-bromo-4-chloro-3-indolyl-D-galactopyranoside (X-gal). The tissue specificity of promoters in transgenic constructs is routinely tested by using a promoter of choice to drive lacZ. Alternatively, the targeted expression of Cre recombinase to perform in vivo recombination of loxP sites can be visualized by beta-gal staining in mice carrying a Cre-activated lacZ transgene, such as the ROSA26 strain. In the course of our investigations, we examined beta-gal activity in bone tissue from genetically normal mice using standard detection methodology and found very high endogenous activity in bone-resorbing osteoclasts. This was true in frozen, paraffin, and glycol methacrylate sections. X-gal staining colocalized with the osteoclast marker, tartrate-resistant acid phosphatase ( TRAP). beta-gal activity was present in osteoclasts in long bones, in the mandible, and in both neonatal and more mature animals. We present this brief article as a caution to those testing genetic models of skeletal gene expression using beta-gal as a marker gene.

Osteoclasts differentiate from hematopoietic precursors under systemic and local controls. Chemokines and receptors direct leukocyte traffic throughout the body and may help regulate site-specific bone resorption. We investigated bone gene expression in vivo during rapid osteoclast differentiation induced by colony-stimulating factor 1 (CSF-1) in Csf1-null toothless (tl/tl) rats. Long-bone RNA from CSF-1-treated tl/tl rats was analyzed by high-density microarray over a time course. TRAP (tartrate-resistant acid phosphatase)-positive osteoclasts appeared on day 2, peaked on day 4, and decreased slightly on day 6, as marrow space was expanding. TRAP and cathepsin K mRNA paralleled the cell counts. We examined all chemokine and receptor mRNAs on the arrays. CCL9 was strongly induced and peaked on day 2, as did its receptor, CCR1, and regulatory receptors c-Fms (CSF-1 receptor) and RANK (receptor activator of nuclear factor kappaB). Other chemokines and receptors showed little or no significant changes. In situ hybridization and immunohistochemistry revealed CCL9 in small, immature osteoclasts on day 2 and in mature cells at later times. Anti-CCL9 antibody inhibited osteoclast differentiation in culture and significantly suppressed the osteoclast response in CSF-1-treated tl/tl rats. While various chemokines have been implicated in osteoclastogenesis in vitro, this first systematic analysis of chemokines and receptors during osteoclast differentiation in vivo highlights the key role of CCL9 in this process.

We identified an EST sequence that was up-regulated during osteoclast formation in the rat. Investigating further, we cloned the cDNA from rat long bone and found it to be highly homologous to the mouse CC chemokine, CCL9, both at the nucleotide and amino acid levels. The rat CCL9 amino acid sequence is 74% identical to the mouse sequence, with an additional 11% similar amino acids. Recombinant rat CCL9 was used in chemotaxis assays of rat bone marrow cells and it was found to have a strong and dose-dependent effect. In addition, CCL9 mRNA was very highly up-regulated during osteoclast differentiation of rat bone marrow-derived mononuclear cells, increasing by over 100-fold when stimulated by colony stimulating factor-1 and the TNF superfamily member, RANKL. Together, these results establish that, like the mouse, the rat also uses CCL9 to promote the complex process of osteoclast formation.

The transition from androgen-dependent to aggressive androgen-independent growth is an important development in prostate cancer, and the identification of genes expressed differentially between these two phenotypes could provide new therapeutic targets as well as prognostic markers. Using a subtractive approach designated linker capture subtraction, we have now isolated several genes expressed differentially between the human prostate carcinoma cell lines LNCaP and PC-3, which are androgen-dependent and androgen-independent, respectively. In addition to 10 known genes that may be involved in signal transduction, tumor growth, tumor invasion, and metastasis, 3 novel genes were isolated. One of these, designated NPG-1, was expressed by the majority of primary tumors examined. Moreover, the degree of NPG-1 expressed correlated with an aggressive histopathology.

Low levels of dietary selenium are associated with increased risk of malignancy of several organs, including the prostate. Using a subtractive approach called linker capture subtraction, we have found that the human selenium-binding protein gene hSP56 is differentially expressed by the relatively slow-growing, androgen-sensitive prostate cancer cell line LNCaP but not by the more rapidly growing androgen-insensitive lines PC-3 and DU145. We confirmed this differential expression by Northern blot analysis. Importantly, hSP56 expression by LNCaP cells was reversibly down-regulated by exogenous androgen in a concentration-dependent manner. Marked differences in steady-state hSP56 mRNA levels were found in a variety of normal and neoplastic human cells that were examined. hSP56 expression was especially high in normal tissues that appear to benefit from the cancer-protective action of dietary selenium and was low in many neoplastic cells. The results suggest that hSP56 may play a role in determining the neoplastic phenotype.

We have developed a simple and effective method, designated linker capture subtraction (LCS), for cloning differentially expressed genes between two cell types or between cells treated in two different ways. In the first step of the method, two mRNA populations are converted to double-stranded cDNAs, fragmented, and ligated to linkers for PCR amplification. In the second step, the linkered DNA (tester) from one mRNA population is hybridized to an excess of the unlinkered DNA (driver) from the other mRNA population, followed by incubation with mung bean nuclease which digests single-stranded DNA specifically. This leaves only tester-tester homohybrids to be amplified by PCR in the following step, so as to achieve an enrichment of tester-specific sequences. The amplified PCR products are then used as tester for another round of subtraction. The process of subtraction is carried out three times, and the final PCR products are inserted into a vector for clonal analysis. We have used the strategy to begin to clone and identify the genes expressed differentially between the human prostate cancer cell lines LNCaP and PC-3, which have different tumorigenic and metastatic potentials. We demonstrated strong enrichment of target sequences. We also report the identities of two of the genes expressed differentially in these cell lines. One is prostate-specific antigen (PSA) which is known to be expressed in LNCaP but not in PC-3. The other is vimentin, the differential expression of which has not been reported previously in these prostate cancer cells.