Dear Federico,

I must say that this is a very interesting question and I also like to say that I do not have a very straight forward answer. As you mentioned depleting calcium either by using EDTA or EGTA in the cell culture is an established method which is directly linked to tight junction and gap junction destabilization pathway. Other complex signaling pathways also exist that works either through cytoskeleton stabilization or PAR-aPKC complex formation that helps cells to maintain its polarity. The alternative approach that came to my mind is siRNA intervention to break the PAR-aPKC  complex formation.

As a ready reference I am sharing an article that could shed more light on this idea.

Hope that helps.

The molecular machinery of how epithelial cells maintain the cellular polarity is a complex issue, so that I would like to focus on hemidesmosome-relared proteins. In the case of symmetrical cell division often observed in proliferative stem cell in the niche, the round co-localization of Rac1 and ColXVII (also named BP180) is thought to be necessary. In contrast, in the case of asymmetrical cell division which provides 1 stem cell and 1 progenitor cell (=relatively differentiated cell), the apical aPKC/Rac1/Par3 localization and the linear-pattern localization of ColXVII at the basement membrane is frequently recognized. Knockdown of ColXVII induces the exhaustion of stem cells particularly in the hair follicle stem cells due to the disruption of the cellular polarity. After all, the abnormal polarity of stem cells fails to maintain the stem cell pool providing the precursor cells for the rapid turn-over and wound-healing in response to tissue damage.

There is a aPKC chemical inhibitors (see below) or could use siRNA to any of the key polarity complexes eg scribble-RNAi.

Biochem J. 2013 Apr 15;451(2):329-42. doi: 10.1042/BJ20121871.

Adenosine-binding motif mimicry and cellular effects of a thieno[2,3-d]pyrimidine-based chemical inhibitor of atypical protein kinase C isoenzymes.

Kjær S1, Linch M, Purkiss A, Kostelecky B, Knowles PP, Rosse C, Riou P, Soudy C, Kaye S, Patel B, Soriano E, Murray-Rust J, Barton C, Dillon C, Roffey J, Parker PJ, McDonald NQ.

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Abstract

The aPKC [atypical PKC (protein kinase C)] isoforms ι and ζ play crucial roles in the formation and maintenance of cell polarity and represent attractive anti-oncogenic drug targets in Ras-dependent tumours. To date, few isoform-specific chemical biology tools are available to inhibit aPKC catalytic activity. In the present paper, we describe the identification and functional characterization of potent and selective thieno[2,3-d]pyrimidine-based chemical inhibitors of aPKCs. A crystal structure of human PKCι kinase domain bound to a representative compound, CRT0066854, reveals the basis for potent and selective chemical inhibition. Furthermore, CRT0066854 displaces a crucial Asn-Phe-Asp motif that is part of the adenosine-binding pocket and engages an acidic patch used by arginine-rich PKC substrates. We show that CRT0066854 inhibits the LLGL2 (lethal giant larvae 2) phosphorylation in cell lines and exhibits phenotypic effects in a range of cell-based assays. We conclude that this compound can be used as a chemical tool to modulate aPKC activity in vitro and in vivo and may guide the search for further aPKC-selective inhibitors.

PMID:

23418854

Reproduction. 2004 Jun;127(6):653-67.

PKC signalling regulates tight junction membrane assembly in the pre-implantation mouse embryo.

Eckert JJ1, McCallum A, Mears A, Rumsby MG, Cameron IT, Fleming TP.

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Abstract

Epithelial differentiation including tight junction (TJ) formation occurs exclusively within the trophectoderm (TE) lineage of the mouse blastocyst. Here we examine mechanisms by which TJ protein membrane assembly might be regulated by protein kinase C (PKC) in the embryo. To overcome the inherent staging asynchrony of individual blastomeres within intact embryos, we have used isolated inner cell masses (ICMs) from early blastocysts to induce epithelial differentiation in their outer cells responding to their new cell contact pattern. Two TJ proteins examined retain their order of membrane assembly in isolated ICMs in culture as during normal development (early-assembling ZO-2 and late-assembling ZO-1alpha(+)), but this process is highly accelerated. Using six chemical modulators of PKC activity, we show here that PKC signalling is involved in the regulation of TJ membrane assembly. While indolactam-mediated PKC activation stimulates membrane assembly of both TJ proteins, TPA-mediated PKC activation stimulates only that of ZO-1alpha(+). The PKC inhibitors Ro-31-8220, Ro-31-8425 and Gö 6983 suppress the stimulatory effect of both PKC activators on membrane assembly to varying extents according to inhibitor and TJ protein examined. Gö 6983 similarly inhibits ZO-2 and ZO-1alpha(+) membrane assembly. PKC inhibition by Gö 6976 appeared to stimulate TJ membrane assembly. Despite the broad PKC isotype specificity of the inhibitors used, these data suggest that the two TJ proteins are differently regulated by PKC isotypes or subfamilies. As Gö 6983 uniquely affects aPKC (particularly PKCzeta) and we find that both PKCdelta and zeta relocate upon activator treatment to colocalise partially with the TJ proteins in isolated ICMs, we suggest that at least PKCdelta and zeta may play a central role in regulating TJ membrane assembly.

Also can try this PKC-zeta inhibitor - PKCζ pseudosubstrate inhibitor (Myristoylated-SIYRRGARRWRKL-OH, Calbiochem), 10 μM.

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Dev Biol. 2013 Mar 1;375(1):13-22. doi: 10.1016/j.ydbio.2013.01.002. Epub 2013 Jan 9.

Atypical PKC, regulated by Rho GTPases and Mek/Erk, phosphorylates Ezrin during eight-cell embryocompaction.

Liu H1, Wu Z, Shi X, Li W, Liu C, Wang D, Ye X, Liu L, Na J, Cheng H, Chen L.

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Abstract

Phosphorylation of Ezrin T567 plays an important role in eight-cell embryo compaction. Yet, it is not clear how Ezrin phosphorylation is regulated during embryo compaction. Here, we demonstrated that inhibition of Mek/Erk or protein kinase C (PKC) signaling reduced the phosphorylation level of Ezrin T567 in eight-cell compacted embryos. Interestingly, the Rho GTPase inhibitor C3-transferase caused basolateral enrichment of atypical PKC (aPKC), as well as basolateral shift of phosphorylated Ezrin, suggesting aPKC may be a key regulator of Ezrin phosphorylation. Moreover, inhibition of PKC, but not Mek/Erk or Rho GTPases, affected the maintenance of Ezrin phosphorylation in compacted embryos. We further identified that aPKC is indeed required for Ezrin phosphorylation in eight-cell embryos. Taken together, Rho GTPases facilitate the apical distribution of aPKC and Ezrin. Subsequently, aPKC and Mek/Erk work together to promote Ezrin phosphorylation at the apical region, which in turn mediates the apical enrichment of filamentous actin, stabilizing the polarized apical region and allowing embryo compaction. Our data also suggested that aPKC might be the Ezrin kinase during eight-cell embryo compaction.

Thanks to all

we are basically trying to establish a good protocol for polarity acquisition in Hepatocellular Carcinoma derived cells in order to understand if dysregulation of signaling involved in polarity are in some way connected to liver carcinogenesis. Only a few cell lines are polarizable (primary hepatocytes and HepG2 are able to form bile canaliculi like structures of cultured in 3d collagen).

I'll try different suggestions from you, I believe that most of them are useful for our purpose. Thanks again