Signaling by fibroblast growth factor (FGF) 18 and FGF receptor 3 (FGFR3) have been shown to regulate proliferation, differentiation, and matrix production of articular and growth plate chondrocytes in vivo and in vitro. Notably, the congenital absence of either FGF18 or FGFR3 resulted in similar expansion of the growth plates of fetal mice and the addition of FGF18 to human articular chondrocytes in culture enhanced proliferation and matrix production. Based on these and other experiments it has been proposed that FGF18 signals through FGFR3 to promote cartilage production by chondrocytes. Its role in chondrogenesis remains to be defined. In the current work we used the limb buds of FGFR3 ؉/؉ and FGFR3 ؊/؊ embryonic mice as a source of mesenchymal cells to determine how FGF18 signaling affects chondrogenesis. Confocal laser-scanning microscopy demonstrated impaired cartilage nodule formation in the FGFR3؊/؊ cultures. Potential contributing factors to the phenotype were identified as impaired mitogenic response to FGF18, decreased production of type II collagen and proteoglycan in response to FGF18 stimulation, impaired interactions with the extracellular matrix resulting from altered integrin receptor expression, and altered expression of FGFR1 and FGFR2. The data identified FGF18 as a selective ligand for FGFR3 in limb bud mesenchymal cells, which suppressed proliferation and promoted their differentiation and production of cartilage matrix. This work, thus, identifies FGF18 and FGFR3 as potential molecular targets for intervention in tissue engineering aimed at cartilage repair and regeneration of damaged cartilage.

Chitosan is a linear cationic biopolymer composed of glucosamine and N-acetyl-glucosamine that is only soluble in acidic aqueous solutions and precipitates when neutralized. However, it was recently discovered that chitosan dissolved in solutions containing glycerol phosphate was soluble at near neutral pH and produced a sol-gel transition when heated. Understanding this unique thermogelling system requires improved characterization of the ionization and solubility behaviors of chitosan, in particular dependencies on temperature, salt, chitosan concentration, and fD, where fD is the fraction of glucosamine monomers (deacetylated monomers) in chitosan. In the current study we performed temperature-controlled titration and dilution experiments on chitosan solutions with fD of 0.72, 0.85, and 0.98 at concentrations ranging from 1.875 to 30 mM of its glucosamine monomer and with 0 to 150 mM added salt. Light transmittance measurements were performed during titration to indicate precipitation. We found the apparent proton dissociation constant of chitosan, pKap, to (1) decrease strongly with increased temperature, (2) increase strongly with increased salt, (3) increase strongly with increased chitosan concentration in low-salt conditions, and (4) decrease weakly with increasing fD. All of the above influences on chitosan pKap were accurately predicted using a mean-field Poisson-Boltzmann (PB) cylindrical cell model where the only adjustable parameter was the temperature-dependent chitosan intrinsic monomeric dissociation constant pK0(T). The resulting chitosan pK0 values at 25 degrees C were in the range from 6.63 to 6.78 for all chitosans and salt contents tested. The temperature dependence of chitosan ionization was found to strongly reduce pK0(T) by 0.023 units per degrees C, for example, resulting in a reduction of chitosan pK0(T) from 7.1 at 5 degrees C to 6.35 at 37 degrees C for fD of 0.72 in 150 mM salt. A similar temperature-dependent reduction of the pKa of the glucosamine monomer was found (-0.027 units per degrees C) while the pKa of glycerol phosphate did not change significantly with temperature. The latter result suggested that chitosan solutions heated in the presence of glycerol phosphate will become partly neutralized by transferring protons to glycerol phosphate and thereby allow attractive interchain forces to form a physically cross-linked gel under the appropriate conditions. Additionally, the degree of ionization of chitosan when it precipitates upon addition of a strong base was measured to be in the range from 0.25 to 0.55 and was found to (1) be insensitive to temperature, (2) increase strongly with increased salt, and (3) increase strongly with fD. The salt effect was accounted for by the PB model, while the influence of fD appeared to be due to acetyl groups impeding attractive chain-to-chain association to increase solubility and require reduced ionization levels to precipitate.

Recently, chitosan dissolved in solutions containing glycerol phosphate (GP) were found to undergo a sol-gel transition when heated and the proposed gelling mechanism was based on increasing hydrophobic interactions with temperature. Subsequently, an investigation of ionization and precipitation behavior of chitosan, including dependencies on temperature, added salt, and fraction of deacetylated monomers (fD) was performed. This latter study revealed important differences in the temperature dependence of pKa of chitosan versus GP and led us to propose an alternative hypothesis for the mechanism of gelation in chitosan-GP systems whereby heat induces transfer of protons from chitosan to glycerol phosphate thereby neutralizing chitosan and allowing attractive interchain forces to form a physical gel. To investigate this specific molecular thermogelling mechanism, temperature ramp experiments on dilute chitosan-GP solutions were performed. Chitosans with fD of 0.72 and 0.98 were used to prepare solutions with a range of molar ratios of GP to chitosan glucosamine monomer of 1.25 to 10 and with 0 or 150 mM added monovalent salt. Light transmittance measurements were performed simultaneously to indicate precipitation in these dilute systems as a surrogate for gelation in concentrated systems. Measured temperatures of precipitation ranged from 15 to 85 degrees C, where solutions with less GP (used in a disodium salt form) had lower precipitation temperatures. A theoretical model using acid-base equilibria with temperature dependent pKa's, including the electrostatic contribution from the polyelectrolyte nature of chitosan, was used to calculate the degree ionization of chitosan (alpha, the fraction of protonated glucosamine monomer) as a function of temperature and showed a significant decrease in alpha with increased temperature due to proton transfer from chitosan to GP. This heat-induced proton transfer from chitosan to GP was experimentally confirmed by 31P NMR measurements during temperature ramp experiments since the chemical shift of 31P of GP is an indicator of its level of protonation. By assuming average temperature independent values of alpha p that were calculated from measured T(p), the model was able to accurately predict measured temperatures of precipitation (T(p)) of all chitosan-GP mixtures. The resulting alpha(p) were temperature independent but increased with increased chitosan fD and with increased salt. Measurements and theory revealed that T(p) can be adjusted in a predictable manner by changing the chitosan-GP molar ratio and thereby systematically tailored to obtain a large range of precipitation temperatures. Finally, similar temperature ramp experiments using inorganic phosphate and MES in place of GP demonstrated that the temperature-induced precipitation of chitosan also occurs with these buffers, confirming that the key feature of the buffer used with chitosan is its ability to absorb heat-stimulated release of chitosan protons and facilitate chitosan neutralization. A theoretical expressio...

Cancer cells exploit the epithelial-to-mesenchymal transition (EMT) program to become metastatic. Cytoskeletal regulators are required in mesenchymal cells where they promote EMT and EMT-induced migration. In a search for regulators of metastasis, we conducted shRNA screens targeting the microtubule plus-end tracking proteins (+TIPs). We show that the +TIP ACF7 is essential both for the maintenance of the EMT program and to promote migration. We find that the E3 ubiquitin ligase HectD1 promotes ACF7-proteasome-mediated degradation. Depletion of HectD1 stabilized ACF7, and this enhanced EMT and migration. Decreased HectD1 expression increased metastases in mouse models and conferred increased resistance to the cytotoxic drug cisplatin. A retrospective analysis of biopsies from breast cancer patients also reveals a correlation between higher ACF7 or lower HectD1 expression with poor clinical outcomes. Together, these results suggest that the control of ACF7 levels by HectD1 modulates EMT and the efficiency of metastasis.

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