Benign cells and calcitonin receptor-negative PC cells were also tested

Benign cells and calcitonin receptor-negative PC cells were also tested. Results MEK or p38 but not JNK reduced CD44 total RNA by 40%C65% in malignancy and benign cells. protein expression. In calcitonin receptor-positive cells only, calcitonin increased CD44 variant RNA and protein by 3 h and persisting to 48 h, apparently dependent on an uninhibited p38 pathway. Cells with constitutive CT expression showed an increase in CD44v7-10 mRNA but a decrease in CD44 total RNA. Conclusion The MEK pathway increases CD44 RNA, while calcitonin, acting through the protein kinase A AZ1 and p38 pathway, facilitates variant splicing. These findings could be used in the formulation of therapeutic methods for PC targeting CD44 alternate splicing. Background CD44, a transmembrane glycoprotein, is the product of a gene that can undergo extensive alternate splicing. The standard (CD44s) isoform is usually ubiquitous but tissue-specific isoforms may include an assortment of 10 variant (v) exons (CD44v). CD44 facilitates multiple cellular functions. CD44 enables cell-cell and cell-matrix adhesion C primarily to its main ligand hyaluronan, and links the cell membrane to the actin cytoskeleton, modulating motility. CD44 is usually universally dysregulated in human malignancy, and this imbalance of isoforms allows tumor growth and invasion [1-8]. CD44v are expressed in prostatic secrectory cells while CD44s is found in the whole epithelium. About 30% of cases of prostate malignancy (PC) undergo a transition from quiescent to aggressive. Altered CD44 and other adhesion molecules permit this transition in which tumor cells detach, interact with proteins that digest stromal matrix, migrate through matrix, and intravasate into lymphovascular channels. By isolating RNA from clinical PC specimens, we discovered that the major variant isoform expressed in PC is CD44v7-10. This PC signature was consistently present in both main and metastatic PC [1-3]. Interference against this CD44v caused a 69% reduction in invasion index compared to untreated control cells[3]. Moreover, PC loses the splicing ability to produce the CD44s expressed in benign prostate[3,9,10]. CD44 must oligomerize to bind matrix ligands or to cause metastasis[11] and variant isoforms, with longer extracellular tails, have altered ability to complex[12]. We found that the CD44v7-10 isoform makes PC cells preferentially bind to fibronectin rather than hyaluronan; re-expression of CD44s causes the predominant ligand to revert from fibronectin back to hyaluronan[4]. In mouse xenografts of PC-3 prostate tumor, forced expression of CD44s reduced growth in vitro and tumorigenicity[5], and our use of RNAi against CD44v7-10 in xenografts yielded comparable effects (unpublished results). In PC, calcitonin (CT) acts as a paracrine growth factor that up-regulates CD44 variant[4,6]. In histologic specimens PC, but not benign secretory epithelium, contains CT[13] and its receptor (CTR)[14], and CT exerts paracrine effects that promote proliferation[15], invasion[16], and metastasis[17]. CTR, essential for prostate malignancy tumorigenicity[18], is coupled to the transduction protein Gs. We have shown that CT promotes alternate splicing leading to CD44v7-10 mRNA and protein[4,6] by acting through Gs signaling[3]. Gs stimulates the cyclic AMP signaling cascade[17,19] and protein kinase A (PKA)[16]. PKA, in turn, acts around the 3 main MAPK pathways: a growth factor-responsive pathway that uses MAP2K (also called MEK) as important downstream AZ1 effector; and two stress-activated pathways, c-jun N-terminal kinase (JNK), and p38 kinase, that respond to stress including cytokines, osmotic shock, and irradiation. CD44 variants activate MAPK pathways[20], sometimes by functioning as co-receptors for growth factors[21]. MAPK pathways, in turn, can cause CD44 alternate splicing to include variant exons[22]. Oncogenes such as ras[7,23] and mitogens using the MEK-ERK MAP kinase (MAPK) pathway[7], but not the p38 pathway[24], induce CD44 promoter activity and increase AZ1 expression of certain CD44v. To test whether these influences modulate RNA levels and alternate splicing of CD44 in PC, we analyzed the Rabbit Polyclonal to NDUFB10 CT signaling system, PKA, and MAPK pathways. CD44 mRNA and protein levels were measured. Methods Cell lines PC-3 cells (American Type Culture Collection, Manassas, VA) were incubated in F12-K medium, 10% fetal calf serum, and antibiotics at 37C in a 5% CO2 incubator. Gs-QL cells, CT+, CT-, and CTR-cells were gifts of Dr. Girish Shah, Univ. of Louisiana-Monroe[17]. The Gs-QL cells were derived from metastasizing PC-3M cells stably transfected by AZ1 a plasmid that directs expression of mutant, constitutively active Gs [17,19]. These three cell.