Jorma A. Määttä

ErbB4 and Its Isoforms: Selective Regulation of Growth Factor Responses by Naturally Occurring Receptor Variants

ErbB4 is a member of the epidermal growth factor receptor (EGFR, ErbB) family that mediates responses to neuregulins and other EGF-like growth factors. ErbB4 is a central regulator of cardiovascular and neural development as well as differentiation of the mammary gland. A role for ErbB4 has also been implicated in malignancies and heart diseases. Four structurally and functionally distinct ErbB4 isoforms have recently been identified. One pair of isoforms differs within their extracellular juxtamembrane domains. These juxtamembrane ErbB4 isoforms are either susceptible or resistant to proteolytic processing that release a soluble receptor ectodomain. Another pair of ErbB4 isoforms differs within their cytoplasmic tails. Analysis of the intracellular signal transduction pathways indicates that both cytoplasmic ErbB4 isoforms can couple to the Shc-MAPK signaling pathway, while the other one is incapable of coupling to the phosphoinositide 3-kinase (PI3-K)-Akt pathway. The differences in the activation of signaling cascades are reflected in the cellular responses stimulated via the cytoplasmic isoforms. Both cytoplasmic ErbB4 isoforms can stimulate proliferation, but the isoform that cannot activate PI3-K is defective in stimulating cellular survival and chemotaxis. Together these four naturally occurring receptor variants provide a new level of diversity to the control of growth factor-stimulated cellular responses. Thus, the ErbB4 isoforms may have distinct and specific roles in the regulation of various developmental and pathological processes.

Angiopoietin-regulated recruitment of vascular smooth muscle cells by endothelial-derived heparin binding EGF-like growth factor

Recruitment of vascular smooth muscle cells (SMC) by endothelial cells (EC) is essential for angiogenesis. Endothelial‐derived heparin binding EGF‐like growth factor (HB‐EGF) was shown to mediate this process by signaling via ErbB1 and ErbB2 receptors in SMCs. 1) Analysis of ErbB‐ligands demonstrated that primary ECs expressed only HB‐EGF and neuregulin‐1. 2) Primary SMCs expressed ErbB1 and ErbB2, but not ErbB3 or ErbB4. 3) Consistent with their known receptor specificities, recombinant HB‐EGF, but not neuregulin‐1, stimulated tyrosine phosphorylation of ErbB1 and ErbB2 and migration in SMCs. 4) Neutralization of HB‐EGF or inhibition of ErbB1 or ErbB2 blocked 70–90% of the potential of ECs to stimulate SMC migration. Moreover, 5) angiopoietin‐1, an EC effector with a role in recruitment of SMC‐like cells to vascular structures in vivo, enhanced EC‐stimulated SMC migration by a mechanism involving up‐regulation of endothelial HB‐EGF. Finally, 6) immunohistochemical analysis of developing human tissues demonstrated that HB‐EGF was expressed in vivo in ECs associated with SMCs or pericytes but not in ECs of the hyaloid vessels not associated with SMCs. These results suggest an important role for HB‐EGF and ErbB receptors in the recruitment of SMCs by ECs and elaborate on the mechanism by which angiopoietins exert their vascular effects.—Iivanainen E., Neli‐markka L., Elenius V., Heikkinen S.‐M., Junttila T. T., Sihombing L., Sundvall M., Määttä J. A., Laine V. J. O., Ylä‐Herttuala S., Higashiyama S., Alitalo K., Elenius K. Angiopoietin‐regulated recruitment of vascular smooth muscle cells by endothelial‐derived heparin binding EGF‐like growth factor. FASEB J. 17, 1609–1621 (2003)

Replicase complex genes of Semliki Forest virus confer lethal neurovirulence.

ABSTRACT Semliki Forest virus (SFV) is a mosquito-transmitted pathogen of small rodents, and infection of adult mice with SFV4, a neurovirulent strain of SFV, leads to lethal encephalitis in a few days, whereas mice infected with the avirulent A7(74) strain remain asymptomatic. In adult neurons, A7(74) is unable to form virions and hence does not reach a critical threshold of neuronal damage. To elucidate the molecular mechanisms of neurovirulence, we have cloned and sequenced the entire 11,758-nucleotide genome of A7(74) and compared it to the highly neurovirulent SFV4 virus. We found several sequence differences and sought to localize determinants conferring the neuropathogenicity by using a panel of chimeras between SFV4 and a cloned recombinant, rA774. We first localized virulence determinants in the nonstructural region by showing that rA774 structural genes combined with the SFV4 nonstructural genome produced a highly virulent virus, while a reciprocal recombinant was asymptomatic. In addition to several amino acid mutations in the nonstructural region, the nsp3 gene of rA774 displayed an opal termination codon and an in-frame 21-nucleotide deletion close to the nsp4 junction. Replacement in rA774 of the entire nsp3 gene with that of SFV4 reconstituted the virulent phenotype, whereas an arginine at the opal position significantly increased virulence, leading to clinical symptoms in mice. Completion of the nsp3 deletion in rA774 did not increase virulence. We conclude that the opal codon and amino acid mutations other than the deleted residues are mainly responsible for the attenuation of A7(74) and that the attenuating determinants reside entirely in the nonstructural region.

Inactivation of estrogen receptor α in bone-forming cells induces bone loss in female mice

The role of the estrogen receptor α (ERα) in bone‐forming cells is incompletely understood at present. To examine the in vivo effects of ERα in these cells, we generated a mouse strain in which the ERα gene is inactivated in osteoblasts via osteocalcin promoter‐regulated cyclic recombinase (Cre) activity (ERαΔOB/ΔOB). This enabled micro‐computed tomography‐ and histomorphometry‐based analysis of ERα‐mediated effects in bone‐forming cells in mice, in which the systemic ERα‐mediated effects are intact. In female ERαΔOB/ΔOB mice, trabecular and cortical bone volumes were significantly reduced (31.5 and 11.4%, respectively) at 3.5 mo of age compared with control ERαfl/fl animals, and their response to ovariectomy was small compared with that of controls. In contrast with females, no differences could be detected in the bone phenotype of young males, whereas in 6‐mo‐old ERαΔOB/ΔOB males, trabecular bone volume (Tb.BV) was decreased (27.5%). The ERα inactivation‐related effects were compared with those of controls having a similar genetic background. Parental osteocalcin‐Cre mice did not show Cre‐related changes. Our results suggest that in female mice, Tb.BV and cortical bone volume are critically dependent on the ERα regulation of osteoblasts, whereas in male mice, osteoblastic ERα is not required for the regulation of bone formation during rapid skeletal growth, but it is involved in the maintenance of Tb.BV.—Määttä, J. A., Büki, K. G., Gu, G., Alanne, M. H., Vääräniemi, J., Liljenbäck, H., Poutanen, M., Härkönen, P., Väänänen, K. Inactivation of estrogen receptor α in bone‐forming cells induces bone loss in female mice. FASEB J. 27, 478–488 (2013). www.fasebj.org

Encephalitogenicity of myelin‐associated oligodendrocytic basic protein and 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase for BALB/c and SJL mice

In search of new encephalitogenic myelin antigens, the 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNP) and 19 000 MW isoform of myelin‐associated oligodendrocytic basic protein (MOBP) were obtained as recombinant proteins by the baculovirus expression system in Spodoptera frugiperda cells and purified to homogeneity by immobilized metal chelate affinity chromatography (IMAC). The purified MOBP was soluble in water and showed retarded migration on sodium dodecyl sulphate–polyacrylamide gel electrophoresis similar to myelin basic protein (MBP). MOBP induced experimental autoimmune encephalomyelitis (EAE) in nine of 15 susceptible SJL OlaHsd mice, causing death in two animals, whereas three of 14 BALB/c mice showed mild symptoms of EAE, manifested as transient weakness of hind limbs. In both mouse strains, periventricular infiltrates of mononuclear cells were observed. In addition, both 46 000 MW and 48 000 MW CNP isoforms were shown to be non‐encephalitogenic for both mouse strains.

Neutrophils secreting tumor necrosis factor alpha infiltrate the central nervous system of BALB/c mice with experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) can be induced in resistant BALB/c mice by ultrasound-formed adjuvant emulsion. In contrast to susceptible mouse strains large numbers of neutrophils secreting TNF-alpha occupied the central nervous system (CNS) of BALB/c mice with severe EAE, whereas only small numbers of macrophages and CD4+ T-cells could be detected. CNS infiltration was preceded with activation of microglial cells. Ultrasound formed adjuvant induced early IFN-gamma expression in popliteal lymph nodes of BALB/c mice, whereas conventional adjuvant induced delayed IFN-gamma production. Although the clinical outcome of EAE was similar to that seen in susceptible mice, the pathogenesis was distinct having possible implications on the different forms seen in multiple sclerosis.

THE SEMLIKI FOREST VIRUS E2 GENE AS A VIRULENCE DETERMINANT

We have determined the nucleotide sequences of the capsid, E3, E2 and 6K genes of the avirulent Semliki Forest virus variant A774 (SFV A7). The sequence analysis revealed a nucleotide identity of 98% for capsid, 98% for E3, 97% for E2 and 98% for 6K genes, as compared with the prototype SFV strain L10. At the protein level, the capsid and E3 polypeptides of SFV A7 both exhibited two amino acid substitutions, whereas point mutations in the 6K gene did not alter the amino acid sequence. In the E2 gene of SFV A7, seven of the 34 point mutations led to an amino acid difference as compared with the L10 strain. Replacement of the E2 glycoprotein gene of the virulent SFV4 clone with the corresponding region of SFV A7 resulted in a new plasmid construct, pME2, that gave rise to infectious virus CME2. CME2 and SFV4 replicated similarly in an immortalized mouse brain cell line (MBA 13). Intraperitoneal injection of 10(6) p.f.u. of CME2 into 4- to 6-week-old BALB/c mice caused mild clinical signs in some mice, whereas the majority of the infected animals remained asymptomatic, similar to infection with the avirulent SFV A7. In contrast, infection with the parental SFV4, a derivative of the virulent L10 strain, was lethal in 80% of mice. Virus titres in blood and brain tissue specimens of BALB/c mice were similar after infection with CME2 or A7 viruses. The results suggest that amino acid differences in the E2 glycoprotein individually or in concert cause the attenuation of CME2.

Physical state of the neuroantigen in adjuvant emulsions determines encephalitogenic status in the BALB/c mouse

A novel form of adjuvant-neuroantigen formulation was established which was highly encephalitogenic in previously resistant BALB/c mice. The antigen formulation contained mouse whole spinal cord homogenate (MSCH), mycobacteria, and mineral oil, identically to the conventional preparation, but emulsification was completed by sonication instead of extrusion. Sonication of MSCH alone did not render a conventionally prepared emulsion encephalitogenic. The novel adjuvant formulation showed reduced water-oil droplet size, and the neuroantigen was located on the surface of the droplets as well as in the intermicellar space, while in the extruded formulation the material was buried in the droplet interior. Mice inoculated with the sonicated emulsion showed strong brain and spinal cord infiltration of lymphoid cells. The sonicated emulsion was highly encephalitogenic in all six BALB/c substrains tested. The results suggest that availability of the neuroantigen is of critical importance for the development of clinical EAE in the BALB/c mouse.

Inactivation of the androgen receptor in bone-forming cells leads to trabecular bone loss in adult female mice

Removal of the androgen receptor (AR) from bone-forming cells has been shown to reduce trabecular bone volume in male mice. In female mice, the role of AR in the regulation of bone homeostasis has been poorly understood. We generated a mouse strain in which the AR is completely inactivated only in mineralizing osteoblasts and osteocytes by breeding mice carrying osteocalcin promoter-regulated Cre-recombinase with mice possessing loxP recombination sites flanking exon 2 of the AR gene (AR(ΔOB/ΔOB) mice). In female AR(ΔOB/ΔOB) mice, the trabecular bone volume was reduced owing to a smaller number of trabeculae at 6 months of age compared with the control AR(fl/fl) animals. In male AR(ΔOB/ΔOB) mice, an increase in trabecular bone separation could already be detected at 3.5 months of age, and at 6 months, the trabecular bone volume was significantly reduced compared with that of male AR(fl/fl) mice. No AR-dependent changes were observed in the cortical bone of either sex. On the basis of micro-computed tomography and histomorphometry, we conclude that in male mice, the AR is involved in the regulation of osteoclast number by osteoblasts, whereas in female mice, the lack of the AR in the bone-forming cells leads to a decreased number of trabeculae upon aging.

Receptor tyrosine kinase inhibition causes simultaneous bone loss and excess bone formation within growing bone in rats.

During postnatal skeletal growth, adaptation to mechanical loading leads to cellular activities at the growth plate. It has recently become evident that bone forming and bone resorbing cells are affected by the receptor tyrosine kinase (RTK) inhibitor imatinib mesylate (STI571, Gleevec®). Imatinib targets PDGF, ABL-related gene, c-Abl, c-Kit and c-Fms receptors, many of which have multiple functions in the bone microenvironment. We therefore studied the effects of imatinib in growing bone. Young rats were exposed to imatinib (150mg/kg on postnatal days 5-7, or 100mg/kg on postnatal days 5-13), and the effects of RTK inhibition on bone physiology were studied after 8 and 70days (3-day treatment), or after 14days (9-day treatment). X-ray imaging, computer tomography, histomorphometry, RNA analysis and immunohistochemistry were used to evaluate bone modeling and remodeling in vivo. Imatinib treatment eliminated osteoclasts from the metaphyseal osteochondral junction at 8 and 14days. This led to a resorption arrest at the growth plate, but also increased bone apposition by osteoblasts, thus resulting in local osteopetrosis at the osteochondral junction. The impaired bone remodelation observed on day 8 remained significant until adulthood. Within the same bone, increased osteoclast activity, leading to bone loss, was observed at distal bone trabeculae on days 8 and 14. Peripheral quantitative computer tomography (pQCT) and micro-CT analysis confirmed that, at the osteochondral junction, imatinib shifted the balance from bone resorption towards bone formation, thereby altering bone modeling. At distal trabecular bone, in turn, the balance was turned towards bone resorption, leading to bone loss.