We recently demonstrated in an immortalized thyroid cell line that integrin stimulation by fibronectin (FN) simultaneously activates two signaling pathways: Ras/Raf/MAPK kinase (Mek)/Erk and calcium Ca2+/calcium calmodulin-dependent kinase II (CaMKII). Both signals are necessary to stimulate Erk phosphorylation because CaMKII modulates Ras-induced Raf-1 activity. In this study we present evidence that extends these findings to normal human thyroid cells in primary culture, demonstrating its biological significance in a more physiological cell model. In normal thyroid cells, immobilized FN-induced activation of p21Ras and Erk phosphorylation. This pathway was responsible for FN-induced cell proliferation. Concurrent increase of intracellular Ca2+ concentration and CaMKII activation was observed. Both induction of p21Ras activity and increase of intracellular Ca2+ concentration were mediated by FN binding to alphavbeta3 integrin. Inhibition of the Ca2+/CaMKII signal pathway by calmodulin or CaMKII inhibitors completely abolished the FN-induced Erk phosphorylation. Binding to FN induced Raf-1 and CaMKII to form a protein complex, indicating that intersection between Ras/Raf/Mek/Erk and Ca2+/CaMKII signaling pathways occurred at Raf-1 level. Interruption of the Ca2+/CaMKII signal pathway arrested cell proliferation induced by FN. We also analyzed thyroid tumor cell lines that displayed concomitant aberrant integrin expression and signal transduction. These data confirm that integrin activation by FN in normal thyroid cells generates Ras/Raf/Mek/Erk and Ca2+/CaMKII signaling pathways and that both are necessary to stimulate cell proliferation, whereas in thyroid tumors integrin signaling is altered.

BackgroundCurrently, the two most commonly used fibrinolytic agents in thrombolytic therapy are recombinant tissue plasminogen activator (rt-PA) and streptokinase (SK). Whereas SK has the advantage of substantially lower costs when compared to other agents, it is less effective than either rt-PA or related variants, has significant allergenic potential, lacks fibrin selectivity and causes transient hypotensive effects in high dosing schedules. Therefore, development of an alternative fibrinolytic agent having superior efficacy to SK, approaching that of rt-PA, together with a similar or enhanced safety profile and advantageous cost-benefit ratio, would be of substantial importance. Pre-clinical data suggest that the novel fibrinolytic recombinant staphylokinase (rSAK), or related rSAK variants, could be candidates for such development. However, since an efficient expression system for rSAK is still lacking, it has not yet been fully developed or evaluated for clinical purposes. This study’s goal was development of an efficient fermentation process for the production of a modified, non-glycosylated, biologically active rSAK, namely rSAK-2, using the well-established single cell yeast Hansenula polymorpha expression system.ResultsThe development of an efficient large scale (80 L) Hansenula polymorpha fermentation process of short duration for rSAK-2 production is described. It evolved from an initial 1mL HTP methodology by successive scale-up over almost 5 orders of magnitude and improvement steps, including the optimization of critical process parameters (e.g. temperature, pH, feeding strategy, medium composition, etc.). Potential glycosylation of rSAK-2 was successfully suppressed through amino acid substitution within its only N-acetyl glycosylation motif. Expression at high yields (≥ 1g rSAK-2/L cell culture broth) of biologically active rSAK-2 of expected molecular weight was achieved.ConclusionThe optimized production process described for rSAK-2 in Hansenula polymorpha provides an excellent, economically superior, manufacturing platform for a promising therapeutic fibrinolytic agent.

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Background: Osteoporosis is a bone disease of the elderly that leads to increased risk of fracture. Currently, it aff ects more than 200 million adults worldwide, placing an enormous economic burden on healthcare providers. Present approved drug treatments of osteoporosis, for example, using bisphosphonates, only reduce bone mineral loss, are variable in prevention of future fractures and can induce serious complications long term. From 2002, availability of the recombinant active, N-terminal 1-34 fragment of human parathyroid hormone 1-34, which directly stimulates bone formation, has provided an alternative, potentially more eff ective therapeutic agent for treating osteoporosis, especially in its most severe form. We have adopted the methylotrophic yeast strain Hansenula polymorpha (H. polymorpha) for production of recombinant parathyroid hormone (rPTH) 1-34 since its expression system is highly inducible and target proteins are secreted intact into the fermentation broth without the need for enzymatic cleavage of a fusion protein. Methods: Culture conditions for the growth of selected clones of H. polymorpha super-transformants and their rPTH production capacity were sequentially optimized through the microtitre plate, shake fl ask, small tank bioreactor scales to the large bioreactor scale. Results: The production of rPTH 1-34 by transformed H. polymorpha was greatly enhanced by adding the calnexin chaperone gene to the expression plasmid. Implementation of fi nal optimized culture medium constituents and operational fermentation conditions, of which maintenance of constant oxygen tension (pO 2) was found to be a critical factor at the 80 litre scale, resulted in yields up to 150 mg active rPTH 1-34 per liter of culture supernatant. Conclusion: A robust fermentation process for the effi cient production of active rPTH 1-34 based on a calnexin super-transformed H. polymorpha clone was developed. Potentially, this has cost-saving benefi ts for the production and marketing of clinical-grade rPTH 1-34.

In some cell types, including a fetal thyroid cell line, denial of adhesion to extracellular matrix induces a type of apoptosis called anoikis. Serum withdrawal in dog and transformed rat thyroid cells also induces programmed cell death. Because serum can stimulate cells to produce some components of the extracellular matrix, it was of interest to determine the role of the matrix in the apoptosis induced by serum withdrawal in normal human thyroid cells in primary culture. The present report demonstrates that thyroid cells selectively produce and deposit insoluble fibronectin (FN) only when stimulated by serum. Adhesion in the presence of serum is dependent upon integrin-FN interaction. Serum withdrawal determines a degradation of the insoluble FN deposited and a detachment of the cells from the plates. In these conditions, cells undergo anoikis, demonstrated by DNA fragmentation and annexin V staining. Apoptosis was prevented by exogenous FN immobilized onto the plates. These results indicate that serum withdrawal induces apoptosis in human thyroid cells, determining FN degradation and loss of cell-matrix adhesion.

Hemophilia A is a X-linked recessive bleeding disorder consecutive to the lack of circulating pro-coagulant factor VIII (FVIII). The most efficient strategy to treat or prevent bleeding in patients with hemophilia A relies on replacement therapy using exogenous FVIII. Commercially available recombinant FVIII are produced using an expensive perfusion technology in stainless steel fermenters. A fed-batch fermentation technology was recently developed to produce 'Neureight', a full-length recombinant human FVIII, in Chinese hamster ovary (CHO) cells. Here, we investigated the structural and functional integrity and lack of increased immunogenicity of Neureight, as compared to two commercially available full-length FVIII products, Helixate and Advate, produced in baby hamster kidney or CHO cells, respectively. Our results demonstrate the purity, stability and functional integrity of Neureight with a standard specific activity of 4235 ± 556 IU/mg. The glycosylation and sulfation profiles of Neureight were similar to that of Advate, with the absence of the antigenic carbohydrate epitopes α-Gal and Neu5Gc, and with sulfation of Y1680, that is critical for FVIII binding to von Willebrand factor (VWF). The endocytosis of Neureight by human immature dendritic cells was inhibited by VWF, and its half-life in FVIII-deficient mice was similar to that of Advate, confirming unaltered binding to VWF. In vitro and in vivo assays indicated a similar immunogenicity for Neureight, Advate and Helixate. In conclusion, the production of full-length FVIII in a fed-batch fermentation mode generates a product that presents similar biochemical, functional and immunogenic properties as products developed using the classical perfusion technology.

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