Integrin Expression on Osteogenic Cells

We had several reasons to select osteogenic cell lines, instead of fibroblasts, for use in our experiments. Most importantly, several cell lines with different cell surface collagen receptor integrin patterns are readily available (all the cell lines mentioned here are available from American Type Culture Collection). For example, MG-63 cells express predominantly a2p1 integrin, whereas a1p1 integrin expression level is very low or entirely miss-ing28,33,43 HOS and Saos-2 cells predominantly express a1 p1 integrin and the expression level of a2p1 integrin is low or can not be detected.35,36,43 However, the HOS cell line has turned out to be problematic because after intensive subculturing, a2p1 integrin levels rise.

Some laboratories seem to have HOS cell lines expressing a2p1 integrin exclusively. By using flow cytometry, Northern blot hybridizations, or immunoprecipitations, we have never been able to detect a2 integrin in Saos-2, even in cells that have been subcultured for several passages.

Originally, MG-63, HOS and Saos-2 cells were derived from osteogenic tumors, more precisely, from osteosarcomas. However, they do not behave like malignant cells and grow in soft agar or generate tumors in nude mice. In cell culture they differentiate under appropriate conditions, but by treating HOS cells with a chemical mutagen, like N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), or exposing them to murine Kirsten sarcoma virus, tumorigenic derivatives of HOS cells are generated (in this case, named HOS-MNNG, and KHOS-NP). These cell lines show, in addition to a1p1 integrin, strongly enhanced of a2p1 integrin35 expression. Two nontumorigenic revertants of KHOS-NP, KHOS-240 and KHOS-312, have moderate expression of both a1p1 and a2p1 integrins.35,36 The link between a2p1 integrin expression and malignant cell phenotype in osteogenic cell lines also raises the question regarding the putative contribution done by a2p1 integrin to the cancer cell behavior. The fact that a2p1 integrin is associated with melanoma progression and invasion makes the function of this integrin even more interesting.34,37

In addition to collagen receptor integrins, all the osteogenic cell lines mentioned above express a3p1 and a5p1 integrins. We have recently analyzed aV integrin expression on Saos-2 cells and they seem to have three aV containing heterodimers: aVp3, aVp5, and aVp6 (Koistinen and Heino, unpublished results).

Distinguishing between the effects of different p1 integrins can be done by blocking their function with specific antibodies. The problem with anti-a chain antibodies is that they rarely are efficient enough. Antibodies against p1 subunit are more potent, but depending on the expression pattern on the cell surface, they usually inhibit several integrin heterodimers. A more efficient method is to alter the cell surface integrin expression by cDNA transfections. We have described a model of integrin heterodimer formation and their maturation as glycoproteins.44,45 According to our observations in a wide variety of cell lines, precursor p1 integrin is produced in large excess over the precursor a subunits. The excess pool of precursor p1 integrin accumulates in the endoplasmic reticulum, where p1 subunit awaits an a subunit to form the a/p complex which allows transport to the Golgi and finally to the cell surface.44 Due to the large intracellular pool of precursor p1 subunit, the overexpression of integrin a subunits alone is often enough for efficient surface expression. However, in cell types that lack an adequate precursor p pool, transfection may alter the expression of other integrins.

To this end, we have successfully transfected a2 integrin subunit to a2 integrin negative HOS and Saos-2 cell lines. A commonly used cytomegalovirus promoter was too weak to drive the expression of integrin subunits, so we instead used a construct carrying a spleen focus forming virus LTR promoter.46 The stable expression of an integrin was achieved by having the neomycin analogue, G418, resistance gene in the same expression construct. The increased expression of a2p1 integrin was assessed by measuring the plasmid derived mRNA with Northern blot analysis and the protein levels with immunoprecipitation. Flow cytometry was used to measure the cell surface expression.

We have also used the antisense approach to downregulate the integrin expression.46 This was accomplished by using the same construct carrying the a2 integrin sequence in an antisense orientation. We found this approach efficient since the surface expression of a2p1 integrin on antisense transfected cells was reduced to 10-50% when compared to the original cells.46 The antisense approach has also successfully reduced the amount of p1 integrin synthesis.47 Our repeated attempts to manipulate the expression of other integrin subunits

Fig.8.2. A schematic picture of in vitro models for collagen fiber reorganization. A. In a floating collagen gel model, the gel is detached from the sides and the bottom of the cell culture well after the gelation is complete. The cells begin to rearrange the collagen fibers until a dense collagen lattice is formed. B. In an anchored collagen gel, the end result is a stressed tissue like structure containing cells with prominent actin stress fibers. Unlike floating gels, the cells plated here continue to synthesize DNA and increase in cell number.

Fig.8.2. A schematic picture of in vitro models for collagen fiber reorganization. A. In a floating collagen gel model, the gel is detached from the sides and the bottom of the cell culture well after the gelation is complete. The cells begin to rearrange the collagen fibers until a dense collagen lattice is formed. B. In an anchored collagen gel, the end result is a stressed tissue like structure containing cells with prominent actin stress fibers. Unlike floating gels, the cells plated here continue to synthesize DNA and increase in cell number.

by the same system have however failed. In general, only a few reports have indicated successful use of antisense strategies in the reduction of integrin expression.

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