NH2-terminal cleavage of xenopus fibroblast growth factor 3 is necessary for optimal biological activity and receptor binding.

Fibroblast growth factor 3 (FGF3) was originally identified as the mouse proto-oncogene Int-2, which is activated by proviral insertion in tumors induced by mouse mammary tumor virus. To facilitate the biological characterization of the ligand, we have analyzed its homologue in Xenopus laevis, XFGF3. Here we confirm that the X. laevis genome contains two distinct FGF3 alleles, neither of which is capable of encoding the NH2-terminally extended forms specified by the mouse and human FGF3 genes. Unlike the mammalian proteins, XFGF3 is efficiently secreted as a Mr 31,000 glycoprotein, gp31, which undergoes proteolytic cleavage to produce an NH2-terminally truncated product, gp27. Processing removes a segment of 18 amino acids immediately distal to the signal peptide that is not present in the mammalian homologues. By inserting an epitope-tag adjacent to the cleavage site, we show that a substantial amount of the gp27 is generated intracellularly, although processing can also occur in the extracellular matrix. Two residues are also removed from the COOH terminus. To compare the biological properties of the different forms, cDNAs were constructed that selectively give rise to the larger, gp31, or smaller, gp27, forms of XFGF3. As judged by their ability to cause morphological transformation of NIH3T3 cells, their mitogenicity on specific cell types, and their affinity for the IIIb and IIIc isoforms of Xenopus FGF receptors, gp27 has a much higher biological activity than gp31. Sequence comparison revealed an intriguing similar cleavage motif immediately downstream of the signal peptide cleavage site in the NH2-terminus of mouse and human FGF3. Analysis of secreted mutant mouse FGF3 confirmed an additional NH2-terminal processing at the corresponding sequence motif. NH2-terminal trimming of Xenopus and mammalian FGF3s may therefore be a prerequisite of optimal biological activity.

Lactoferrin expression in mammary epithelial cells is mediated by changes in cell shape and actin cytoskeleton.

Lactoferrin is a secreted iron binding protein which is expressed during normal functional development of mammary epithelium. Murine mammary epithelial cell lines competent for milk protein expression were used to identify microenvironmental factors that regulate lactoferrin expression. While lactoferrin was not expressed in adherent monolayer cultures under standard subconfluent conditions on plastic, lactoferrin mRNA and protein steadily accumulated when the cells aggregated to form spheroids on a reconstituted basement membrane gel. However, unlike other milk proteins such as beta-casein, lactoferrin expression was also induced at high cell density in the absence of exogenously added basement membrane or prolactin. These results led us to examine whether changes in cell growth, cell-cell interactions and/or cell shape were responsible for regulation of lactoferrin gene expression. Rounded, non-proliferating cells in suspension in serum-free medium expressed lactoferrin even as single cells. Conversely, lactoferrin expression could be inhibited in non-proliferative cells in serum-free medium by maintaining them in contact with an air-dried extracellular matrix which caused the cells to retain flat, spread morphologies. These findings indicated that cessation of cell growth was not sufficient, that cell-cell interactions were not required, and that cell culture conditions which minimize cell spreading may be important in maintaining lactoferrin expression. Additional data supporting this latter concept were generated by treating spread cells with cytochalasin D. The resulting disruption of microfilament assembly induced both cell rounding and lactoferrin expression. Shape-dependent regulation of lactoferrin mRNA was both transcriptional and post-transcriptional. Surprisingly, treatment of rounded cells with a transcription inhibitor, actinomycin D, produced a stabilization of lactoferrin mRNA, suggesting that transcription of an unstable factor is required for degradation of lactoferrin mRNA. Importantly, lactoferrin mRNA expression was regulated similarly in early passage normal human mammary epithelial cells. In vivo, the changing extracellular matrix components of the mammary gland during different stages of normal and abnormal growth and differentiation may provide different physical constraints on the configurations of cell surface molecules. These physical constraints may be communicated to the cell interior through mechanical changes in the cytoskeleton. Unlike beta-casein whose expression is upregulated by specific integrin-mediated signals, lactoferrin may be representative of a class of proteins synthesized in the mammary gland using basal transcriptional and translational machinery. The suppression of lactoferrin expression that is observed in monolayer culture and in malignant tissues may reflect inappropriate cell shapes and cytoskeletal structures that are manifested under these conditions.

FGF3 from Xenopus laevis.

Fibroblast growth factor 3 (FGF3) was first identified as the product of a cellular oncogene activated by mouse mammary tumour virus but its normal role appears to be in the developing embryo. To gain further insights into its function, we have isolated sequences encoding the FGF3 homologue in Xenopus laevis, XFGF3. COS-1 cells transfected with XFGF3 cDNA express a 31 kDa product, p31, generated by signal peptide cleavage and Asn-linked glycosylation at the single consensus site. This product is secreted and becomes associated with the cell surface and extracellular matrix. Proteolytic cleavage of p31 in the extracellular compartment results in an amino-terminally truncated product, p27, that is also glycosylated. Both p31 and p27 bind quantitatively to heparin-Sepharose and can be displaced from the cell surface and extracellular matrix by soluble heparin. Conditioned medium containing these two proteins is capable of inducing transient morphological transformation of NIH3T3 cells and of stimulating DNA synthesis in quiescent C57MG and BALB/MK cells which express different isoforms of FGF receptors 1 and 2. Since XFGF3 behaves very differently from its mouse counterpart, we constructed chimeras in which amino-terminal sequences from XFGF3 were fused with carboxy-terminal sequences from mouse FGF3. Increasing the contribution from mouse FGF3 led to a more restricted host range for the chimeric ligand.

Developmental expression of the Xenopus int-2 (FGF-3) gene: activation by mesodermal and neural induction.

We have used a probe specific for the Xenopus homologue of the mammalian proto-oncogene int-2 (FGF-3) to examine the temporal and spatial expression pattern of the gene during Xenopus development. int-2 is expressed from just before the onset of gastrulation through to prelarval stages. In the early gastrula, it is expressed around the blastopore lip. This is maintained in the posterior third of the prospective mesoderm and neuroectoderm in the neurula. A second expression domain in the anterior third of the neuroectoderm alone appears in the late gastrula, which later resolves into the optic vesicles, hypothalamus and midbrain-hindbrain junction region. Further domains of expression arise in tailbud to prelarval embryos, including the stomodeal mesenchyme, the endoderm of the pharyngeal pouches and the cranial ganglia flanking the otocyst. It is shown, by treatment of blastula ectoderm with bFGF and activin, that int-2 can be expressed in response to mesoderm induction. By heterotypic grafting of gastrula ectoderm into axolotl neural plate, we have also demonstrated that int-2 can be expressed in response to neural induction. These results suggest that int-2 has multiple functions in development, including an early role in patterning of the anteroposterior body axis and a later role in the development of the tail, brain-derived structures and other epithelia.