Matrix biology

Characterization of these involves: epitope mapping from domain down to amino acid level, competitive ELISAs, and various functional assays on human cells involved in tissue and tumor fibrosis. Epitope mapping involves generating deletion constructs of integrin alpha11, expression in HEK 293 cells and Western blotting as read-out assay.

The alpha11 antibodies are also used to characterize alpha11 expression in normal and pathological human tissues and cells

We have generated a novel transgenic strain for future conditional gene deletion in an alpha11 integrin specific manner. 3kb of the human integrin alpha11 promoter¹ has been linked to Cre-recombinase and inserted via integrase method into one specific locus. The ITGA11-Cre mouse has been crossed with R26R lacZ reporter mice².

Characterization involves X-gal staining of embryonic and adult tissues, as well as hypertrophic hearts and wounded skin, and isolation of cells from transgenic mouse to relate expression to fibroblast heterogeneity. Once characterization is complete ITGA11-Cre will be mated with a mouse strain with a loxP flanked gene for proof of concept that ITGA11-Cre can delete genes in an a11-specific manner in vivo.

Ongoing studies focus on the role of the integrin alpha11 cytoplasmic tail and the alpha11 I domain.

- In one set of experiments evolutionary conserved residues have been used to pinpoint functionally important residues in the cytoplasmic tail. Mutated alpha11 integrin cDNAs have been stably transfected into cells lacking other collagen receptors and used to test functionality in cell adhesion, collagen reorganization, spheroid invasion and proliferation assays.

- In another set of ongoing experiments chimeric integrins have been generated combining different regions of alpha2 and aalpha11 integrins to elucidate role of integrin alpha11 in fibrillar adhesions. A novel fibrillogenesis assay using fluorescently labeled collagen and microscopic recording of assembly of fluorescent collagen molecule bs is used to monitor fibrillogenesis.

- In an attempt to identify interacting partners for integrin alpha11 cytoplasmic tail proximity ligation approaches³ are being established using biochemical and proteomic methods.

Recent studies in experimental tumors have demonstrated increased tumor stiffness in areas with high stromal expression of integrin alpha11¹. This has prompted us to test the effect of mechanical strain on integrin alpha11 expression using 2D collagen I linked polyacrylamide gels of different stiffness.

To validate obtained mechanoregulation results, experiments will be repeated in different cell types and performed under 3D conditions using attached and floating collagen I gels as previously done by our group⁵.

Further experiments involve combining stiffness experiments with hypoxic conditions and different growth factors to look how these cooperate with mechanoregulation. This work will include use of fibroblasts deficient in certain growth factor receptors and rescue experiments where constitutively activated receptors are used to rescue knockout phenotype under conditions of different substrate stiffness.

Finally, the signaling basis for integrin alpha11 mechanoregulation will be investigated.

1. Lu, N. et al. The human alpha11 integrin promoter drives fibroblast-restricted expression in vivo and is regulated by TGF-beta1 in a Smad- and Sp1-dependent manner. Matrix Biol. 29, 166-176 (2010).

2. Soriano, P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat. Genet. 21, 70-71 (1999).

3. Roux, K.J., Kim, D.I., Raida, M. & Burke, B. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J. Cell Biol. 196, 801-810 (2012).

4. Navab, R. et al. Integrin alpha11beta1 regulates cancer stromal stiffness and promotes tumorigenicity and metastasis in non-small cell lung cancer. Oncogene 35, 1899-1908 (2016).

5. Carracedo, S. et al. The fibroblast integrin alpha11beta1 is induced in a mechanosensitive manner involving activin A and regulates myofibroblast differentiation. J. Biol. Chem. 285, 10434-10445 (2010).