The Hippo pathway in skeletal muscle: a self-interview

This blog entry is work in progress and I will correct or update it once I note errors or find more relevant information.  Q question, A answer.

Q What is the Hippo pathway?

A The Hippo pathway is a near-ubiquitously expressed signal transduction pathway which potently regulates the function and identity of embryonic and adult stem cells (Tremblay & Camargo, 2012), organ size (Pan, 2007), specific functions in adult organs and its dysregulation causes cancer (Harvey et al., 2013).

Q Why is it called ‘Hippo pathway’?

A Apparently the leading researchers in the field phoned each other and agreed on the suggestion by Georg Halder to name the pathway ‘Hippo pathway’ based on the name of one of the Hippo pathway kinases in the fly (information picked up at the bar during the second Hippo workshop in Rome...).

Q How does it work?

A The Hippo pathway is a mushrooming network of signalling proteins which can be sub-divided into the canonical Hippo pathway (Mst1/2 and Lats1/2 are the two canonical kinases), and non-canonical Hippo signalling mechanisms such as mechanotransduction which was shown by Stefano Piccolo's group (Dupont et al., 2011) and signalling via G protein-coupled receptors which was linked to the Hippo pathway by Kun-Liang Guan's team (Yu et al., 2012). Canonical and non-canonical Hippo signalling together regulate the activity of the transcriptional co-factors Yap (Huang et al., 2005;Sudol, 1994;Sudol et al., 1995) and Taz (Kanai et al., 2000) (Vglls are related factors). Yap and Taz can co-activate several transcriptional co-factors of which the Tead transcription factors are the most important ones (Zhao et al., 2008). Tead transcription factors bind to muscle C, A and T (Mar & Ordahl, 1988;Yoshida, 2008) CATTCC DNA motifs. Note that the MCAT elements are identical to GTIIC (Davidson et al., 1988) and Hippo response elements (Wu et al., 2008). Also sometimes the sequence of the other strand is given and no, this is not a plot to confuse non-Hippo reearchers. 

Figure 1. Hippo pathway. In the canonical Hippo pathway the kinases Mst1/2 and Lats1/2 inhibit the transcriptional co-factors Yap and Taz especially by phosphorylation of Ser127 and Ser89, respectively. Yap and Taz co-activate Tead1-4 and other transcription factors. Tead transcription factors regulate gene expression by binding to so-called muscle C, A, and T (MCAT) motifs which have a CATTCC DNA sequence motif. Such MCAT motifs are found in the promoters of many regulatory and functional skeletal muscle genes. Yap and Taz can also be regulated by a plethora of other mechanisms including mechanosensors (exericse physiologists should listen up) and agents such as adrenaline (again this should interest exercise physiologists) which signal via G protein-coupled receptors. This is termed the non-canonical Hippo pathway.

Q If it is so important, why is it only studied now?

A Good question, next question. Also the question is incorrect as the elements of the Hippo pathway were studied since the late 1980s but the real importance for mammals became only apparent when it was shown by Duojia Pan, Fernando Camargo and Rudolf Jaenisch that an over expression of constitutively active Yap increased liver size by ≈4-fold (Dong et al., 2007;Camargo et al., 2007). Currently Hippo pathway research is expanding at a great rate as it seems to have major roles in almost all organs.

Q How was it discovered?

A The discovery of the Hippo pathway can be traced back to two strands of research.

Strand 1: The major discoveries were made by searching for tumour suppressor genes in the fly (Harvey & Tapon, 2007). The knockout of such tumour suppressor genes by Nic Tapon, Kieran Harvey, Iswar Hariharan, Georg Halder, Duojia Pan and others led to organ overgrowth due to increased proliferation and reduced apoptosis. This way the canonical Hippo pathway was discovered. Later it was shown that the canonical Hippo pathway worked by suppressing the activity of the transcriptional co-factors Yap and Taz (Huang et al., 2005). The final link to transcription was made by showing that Yap and Taz co-activate mainly Tead transcription factors (Zhao et al., 2008).

Strand 2: Several important discoveries were made by muscle-focussed researchers who found already in 1988 that the Hippo pathway-targeted MCAT motif was required for the expression of cardiac troponin T in skeletal muscle (Mar & Ordahl, 1988). Janet Mar, Charles Ordahl, Iain Farrance, Irwin Davidson, Alexandre Stewart, Thomas Braun and their groups then studied the regulation of these MCAT elements and characterised Teads, Vglls but not Yap and Taz which are the most potent Hippo  downstream pathway members. Yap was first characterised by Marius Sudol (Sudol, 1994;Sudol et al., 1995). This shows that muscle was on the Hippo radar even before the Hippo pathway was recognised and named as such.

Q What is the function of the Hippo pathway in skeletal muscle?

A We have shown that all major elements of the Hippo pathway are expressed in skeletal muscle (Watt et al., 2010). Moreover, genes with functionally important MCAT elements include α-actin, type I myosin heavy chain and several other genes that regulate muscle development or myogenesis (Yoshida, 2008). We found that Yap is generally active in activated satellite cells (Judson et al., 2012) and myoblasts (Watt et al., 2010) (proliferating muscle cells) and becomes deactivated by transcriptional down-regulation and increased inhibitory Ser127 phosphorylation when these cells fuse to differentiate into muscle fibres. We also found that the expression of activated Yap drives the proliferation but inhibits the differentiation of myoblasts and activated satellite cells which is consistent with the function of Yap in other organs (Judson et al., 2012). Thus active Yap increases satellite cell numbers.

When over expressing Yap in adult muscle (not including satellite cells) then the muscle deteriorates, muscle fibres become nectrotic and some fibres resemble fibres found in patients with centronuclear dystrophy (Judson et al., 2013). This is a puzzling finding but suggests to us that Yap does not co-activate MCAT elements in genes such as α-actin and type I myosin heavy chain which are required for the normal function of differentiated skeletal muscle. There are caveats though as Yap may only be activated briefly in differentiated muscle.

Another study suggests that Tead1 over expression in adult muscle leads to a partial fast-to-slow fibre type shift (Tsika et al., 2008). However, a careful interpretation of this finding is needed as Tead transcription factors without co-factor activation generally repress genes (Koontz et al., 2013).

Q. Phew, that was a lot. Can you just summarise?

A. Of course. The Hippo pathway comprises the canonical Mst1/2-Lats1/2 kinases and the non-canonical pathway includes mechanotransduction and signalling via G-protein coupled receptors. The Hippo pathway is expressed in skeletal muscle and has incompletely understood functions in satellite cells and differentiated muscle fibres. In satellite cells Yap becomes activated during satellite cell activation and when active it increases satellite cell numbers by promoting their proliferation.

The role in differentiated muscle is incompletely understood. Key differentiated muscle genes such as α-actin and type I myosin heavy chain have MCAT elements in their regulatory regions and should be responsive to Hippo signalling. However, our results suggest that high Yap activity is detrimental which does not exclude that short periods of Yap activation maydrive the expression of key genes in differentiated muscle. Thus it is incompletely understood how MCAT motifs in key muscle genes are targeted and whether the Hippo pathwa is responsive to exercise or nutrition or other stimuli. Still plenty of work to do!

Q. Is there a review on the Hippo pathway in skeletal muscle?

A. We are currently writing one. I’ll keep you posted.  

Reference list

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